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J_Muscle_Res_Cell_Motil-3-1-2045119 | Conduction velocities in amphibian skeletal muscle fibres exposed to hyperosmotic extracellular solutions
| Early quantitative analyses of conduction velocities in unmyelinated nerve studied in a constantly iso-osmotic volume conductor were extended to an analysis of the effects of varying extracellular osmolarities on conduction velocities of surface membrane action potentials in Rana esculenta skeletal muscle fibres. Previous papers had reported that skeletal muscle fibres exposed to a wide range of extracellular sucrose concentrations resemble perfect osmometers with increased extracellular osmolarity proportionally decreasing fibre volume and therefore diminishing fibre radius, a. However, classical electrolyte theory (Robinson and Stokes 1959, Electrolyte solutions 2nd edn. Butterworth & Co. pp 41–42) would then predict that the consequent increases in intracellular ionic strength would correspondingly decrease sarcoplasmic resistivity, Ri. An extension of the original cable analysis then demonstrated that the latter would precisely offset its expected effect of alterations in a on the fibre axial resistance, ri, and leave action potential conduction velocity constant. In contrast, other reports (Hodgkin and Nakajima J Physiol 221:105–120, 1972) had suggested that Riincreased with extracellular osmolarity, owing to alterations in cytosolic viscosity. This led to a prediction of a decreased conduction velocity. These opposing hypotheses were then tested in muscle fibres subject to just-suprathreshold stimulation at a Vaseline seal at one end and measuring action potentials and their first order derivatives, dV/dt, using 5–20 MΩ, 3 M KCl glass microelectrodes at defined distances away from the stimulus sites. Exposures to hyperosmotic, sucrose-containing, Ringer solutions then reversibly reduced both conduction velocity and maximum values of dV/dt. This was compatible with an increase in Ri in the event that conduction depended upon a discharge of membrane capacitance by propagating local circuit currents through initially passive electrical elements. Conduction velocity then showed graded decreases with increasing extracellular osmolarity from 250–750 mOsm. Action potential waveforms through these osmolarity changes remained similar, including both early surface and the late after-depolarisation events reflecting transverse tubular activation. Quantitative comparisons of reduced-χ 2 values derived from a comparison of these results and the differing predictions from the two hypotheses strongly favoured the hypothesis in which Riincreased rather than decreased with hyperosmolarity.
Introduction
A classic paper by Hodgkin (1954) (see also: Adrian 1975; Noble 1979) performed a quantitative cable analysis of the local circuit currents thought responsible for action potential propagation in unmyelinated nerve fibres studied in large volumes of constantly iso-osmotic extracellular fluid and demonstrated that their conduction velocities should vary as the square root of fibre radius, a. Action potential conduction velocity is also physiologically important in striated muscle: it ensures a rapid, sarcomeric activation leading to near-synchronous muscle contraction. The present paper accordingly extends these early analyses to effects of varying extracellular osmolarity on conduction velocities of surface membrane action potentials in Rana esculenta skeletal muscle. Skeletal muscle membrane differs from unmyelinated nerve in including an excitable transverse (T-) system whose activation initiates muscle contraction (Adrian et al. 1969, 1970; Huang 1993; Nielsen et al. 2004; Stephenson 2006). Nevertheless, it is likely that the initial sarcolemmal membrane activation precedes the subsequent inward tubular excitation and that the latter may take place partially independently of the propagation of the surface excitation wave. Sheikh et al. (2001) suggested a partially separable T-tubule excitation initiated by Na+ channels selectively clustered around the mouths of the T-tubular lumina: detubulation produced by osmotic shock left surface membrane conduction velocities unchanged. Furthermore, increases in extracellular osmolarity did not alter the tubular diameters at the necks of the T-tubular system important in electrical connectivity between T-tubule network and remaining extracellular space (Launikonis and Stephenson 2002, 2004).
However, variations in surface conduction velocities in skeletal muscle studied under varying osmolarities would be expected to differ from those of nerve fibres studied under constant, iso-osmotic conditions owing to the consequent changes in their cell volumes and fibre diameters. Nevertheless, skeletal muscle volume exhibits close to ideal osmotic behaviour, varying inversely with changes in extracellular osmolarity that would in turn predictably increase solute concentration. The Results section of this paper accordingly first develops the original treatment (Hodgkin 1954; Adrian 1975; Noble 1979) for situations in which fibre diameter is varied by alterations in extracellular osmolarity that would in turn alter intracellular ionic strength. This provided quantitative expectations for any resulting change in conduction velocity that corresponded to two contrasting situations. First, classical electrolyte theory (Robinson and Stokes 1959; Atkins 1998) would predict that increased intracellular ionic strength should proportionally decrease sarcoplasmic resistivity Ri. Our analysis then indicated that this would precisely offset any expected changes in fibre axial resistance, ri, produced by the osmotically induced alterations in fibre diameter and thereby leave conduction velocity unchanged. Secondly, Hodgkin and Nakajima (1972) reported that Ri increased with extracellular osmolarity, possibly due to increases in myoplasmic viscosity, although we certainly do not exclude possible factors arising from the more complex membrane structures found in muscle as opposed to nerve (see e.g. Sheikh et al. 2001). This led to predictions that conduction velocity would decrease with increasing extracellular osmolarity.
These predictions were then investigated by experimental determinations of conduction velocity obtained from action potential records derived from microelectrode measurements made at known distances from defined stimulation sites in surface muscle fibres of Rana esculenta studied at a fixed (∼7°C) temperature at varied extracellular osmolarities. These findings demonstrated reversible changes in the conduction velocities of action potentials the nature of whose waveforms, including early surface and later tubular components, were otherwise unchanged, and distinguished between hypotheses through the observed dependence of conduction velocity on extracellular osmolarity.
Materials and methods
Cold-adapted frogs, Rana esculenta, were killed by concussion followed by pithing (Schedule I: Animal Procedures Act, Home Office, UK). The skin was removed and the tendon of insertion of the sartorius into the patella ligated, cut distally and dissected along its borders up to and including its origin at the pelvic girdle and acetabulum. This was performed at room temperature (21.7 ± 0.51°C; mean ± standard error of the mean; n = 15) in standard Ringer solution consisting of (mM): 115 NaCl, 2.5 KCl, 1.8 CaCl2, 3 Hepes, titrated to pH 7, osmolarity 250 mOsm. The muscle was then stretched to 1.4 times its in situ length and secured in a transilluminated methacrylate polymer (Perspex) chamber by pinning the cleaned acetabulum and the ligature to minimize contractile artefact, which became less evident in solutions of higher osmolarities, and entirely absent with the higher (>350−400 mM) sucrose concentrations. The isolated sartorius muscles had an in situ length of 30.6 ± 0.94 mm and a length of 41.6 ± 1.15 mm (n = 15) when stretched. The muscle was laid over a supporting ramp with its dorsal surface uppermost.
A watertight Perspex partition, coated with a layer of Vaseline, with the muscle running through a notch at its lower surface electrically isolated the recording chamber into two compartments. The only path conducting the brief voltage stimuli applied from two platinum electrodes at opposite ends of the chamber was therefore through the muscle across the partition. The platinum electrode on the side of the chamber containing the acetabulum was the cathode. The temperature of the bath solution was lowered by circulating distilled water cooled in a magnetically stirred ice bath. The water was circulated through a glass coil placed in the chamber in close proximity to the muscle, using a Minipuls 3 peristaltic pump (Gilson, France). A digital thermometer incorporating a remote thermistor (P. Frost, Department of Physiology, Development and Neuroscience, Cambridge), previously linearised and calibrated against a platinum film resistor was placed in the bathing solution near the muscle to allow the temperature to be continuously monitored and adjusted to remain constant throughout the recordings. The temperature was kept constant at 7.0 ± 0.03°C (n = 15) (Sheikh et al. 2001). Action potentials are temperature-sensitive so this allowed conditions to be optimised. Cooling also prolonged fibre viability especially in the hyperosmotic conditions.
The muscle was studied in the range of extracellular osmolarities from 250 to 750 mOsm. Solutions of different osmolarities were made by adding to the standard Ringer solution the membrane-impermeant solute sucrose at varying (150, 250, 350, 400, 450, 500 mM respectively) concentrations to yield solutions whose increased osmolarities (mOsm) were calculated from their total solute concentrations, and checked against measurements using a standard vapour pressure osmometer. Hepes was obtained from Sigma-Aldrich (Gillingham, Dorset, UK) and all other reagents from BDH Limited (UK). Solutions were changed, avoiding contact with the muscle, between tests using a vacuum pump (Edwards, UK) after which approximately 5–10 min elapsed before recording to permit time for both temperature (7°C) and osmotic equilibration; previous reports (Ferenczi et al. 2004) have indicated that 200 s sufficed to permit complete volume changes in response to hyperosmotic solutions.
3 M KCl-filled glass capillary microelectrodes (resistances 5–20 MΩ; tip potentials <5 mV: Adrian 1956) drawn from 1.2 mm (inner diameter) glass tubing were used to obtain membrane potentials. These were mounted via Ag/AgCl junctions to a high impedance voltage follower balanced by matching Ag/AgCl junctions earthing the bath. Only surface fibres showing clear-cut penetrations and stable resting membrane potentials were studied. Rates of rise and fall, dV/dt, in the voltage traces, were obtained by electrical differentiation of the output, with voltage and dV/dt channels filtered between cutoffs of 0 Hz/5 kHz and 0 Hz/20 kHz respectively. Action potentials were elicited by direct just-suprathreshold stimulation which varied between 1.5 and 5 V, to minimise electrotonic spread of the stimulus voltage and the number of fibres in which electrical activity was initiated, across the Perspex partition, through two platinum stimulating electrodes.
Conduction velocity was calculated by dividing the distance from the site of action potential initiation to the position of the recording microelectrode tip, ascertained by a Vernier scale, which gave measurements in cm to two decimal places, by the latency given by the time from a clear stimulus artefact generated to the peak of the dV/dt input, which will be referred to as the maximum dV/dt. The corresponding resting membrane potential, action potential overshoot and the value of maximum dV/dt were also noted. These relatively early events in the timecourse of the regenerative response often could be measured even in records showing small contractile artefact, which often only became evident at later times in the recorded traces.
Results
The experiments utilized an extension of early quantitative analyses (Hodgkin 1954; Adrian 1975) of conduction velocity in unmyelinated nerve studied in a constantly iso-osmotic volume conductor to changes that would result from varying the extracellular osmolarities in Rana esculenta skeletal muscle fibres. The initial analysis used the cable equation for the current density through any patch of flat membrane, expressed as current per unit membrane area,
This describes conduction in a cable whose own finite internal volume is small compared to that of the extracellular conducting fluid in which it is studied to give large ratios between longitudinal intracellular, ri, and extracellular, ro, resistances to current flow per unit length, i.e., ro << ri, permitting extracellular terms relating to ro to be dropped.
Equation (1) is combined with the expression for conduction velocity, using the chain rule of differentiation, to give:where Vm is the internal potential across the membrane at distance x along the fibre. Conduction at constant velocity through local circuit spread of excitation then requires Im to be a single valued function of Vm. The term containing the intrinsic membrane parameters consisting of the area of membrane per unit length of fibre s, and the axial cytoplasmic resistance per unit length ri, is then constant, whence , and where the constant k only depends on passive local membrane electrical properties. Thus:
For unmyelinated cylindrical fibres for which the axial cytoplasmic resistance per unit length, ri, is related to fibre radius a and sarcoplasmic resistivity Ri by Ri = riπ a2, the total fibre volume, vol, = π a2L where L is the length of the fibre and for which the total surface area of the fibre, A = sL:
In the case of skeletal muscle fibres exposed to solutions of different osmolarities, osm, the volume, vol, behaves as a perfect osmometer following the relationship: and reductions in fibre volume due to increased extracellular osmolarity would correspondingly increase solute concentration. Classical electrolyte theory (Robinson and Stokes 1959) then predicts that each participating ion contributes a specific conductance Ksp determined by its solute concentration ηC and a constant Λ defined for any given ion in any specified solute and referred to the conductivity of the ion at a 1 M concentration:
The overall conductivity of such an ideal solution is then the algebraic sum of the conductivities of individual component ions. Decreases in fibre volume resulting from increased extracellular osmolarities then would increase intracellular ionic strength and in turn increase Ksp and proportionally decrease Ri: Equation (7) then gives: Equations (5) and (8) then give,thereby predicting a constant conduction velocity in view of the fact that the terms k′, resulting from the additional proportionalities above, and A are both constants.
In contrast, Hodgkin and Nakajima (1972) suggested that sarcoplasmic resistivity, Ri, increased with increasing extracellular osmolarity, possibly owing to an increased myoplasmic viscosity, with reductions in fibre volume in hyperosmotic solutions, reporting a linear increase in Ri with increasing extracellular osmolarity:where D and E are constants.
From (5) and (10):
Because k′ and A are constants, we have:where D′ and E′ are also constants. This forms a contrasting expectation in which θ is expected to decrease with increasing extracellular osmolarity in a relationship modelled by Eq. (12) as a result of an increase in Ri. The alternative hypotheses could then be quantitatively tested by measurements of θ under different conditions of extracellular osmolarity.
Reversible effects of hyperosmotic extracellular solutions on action potential waveforms and latencies
Figure 1 shows typical action potential and dV/dt waveforms obtained at a temperature of ∼7°C before, during and after exposures to solutions with increased extracellular osmolarity with column A showing the action potential waveforms and column B their corresponding dV/dt records. Panel a shows typical results from fibres in iso-osmotic Ringer at 7°C. The muscle was then exposed to a hyperosmotic (600 mOsm) Ringer solution and recordings resumed 5–10 min following this solution change (Panel b). Finally, the muscle was then returned to iso-osmotic Ringer and recordings again resumed 5–10 min after the solution was restored (Panel c). Latencies were measured from the clear stimulus artefact to the maximum point of the dV/dt trace as seen in column B: this approach provided more consistent measurements of action potential latency than using arbitrarily chosen points on the action potential traces (see also Sheikh et al. 2001).Fig. 1Typical action potential (A) and dV/dt traces (B) obtained from muscle fibres exposed successively to (a) iso-osmotic Ringer (b) hyperosmotic (600 mOsm) Ringer and (c) returned to iso-osmotic Ringer, in the same sartorius muscle
The action potential traces showed prolonged positive after-depolarisation phases lasting well beyond 20 ms following the surface action potential deflections (Fig.1Aa), consistent with persistent excitation of an intact transverse (T-) tubular system (Adrian and Peachey 1973). These persisted both in the hyperosmotic solution (Fig. 1Ab) and the iso-osmotic solution to which the fibres were finally returned (Fig. 1Ac), confirming a persistence in transverse tubular excitation and its excitation following initiation of the surface component of the action potential through these manipulations.
Use of 600 mOsm as opposed to standard Ringer increased the action potential latencies and consequently the calculated values of conduction velocity. Thus, conduction velocities in the initial iso-osmotic Ringer solution, then at 600 mOsm-Ringer solution and finally, the returned iso-osmotic Ringer were 0.98 ± 0.092 m s−1 (n = 11 fibres), 0.58 ± 0.051 m s−1 (n = 10) and 0.72 ± 0.101 m s−1 (n = 6) respectively. The effect of hyperosmolarity on conduction velocity was thus at least partly reversible. However, this decrease in conduction velocity with increased osmolarity (from 250 to 600 mOsm) was not accompanied by qualitative changes in action potential or dV/dt waveform. Likewise, the waveforms showed no qualitative changes following return to the iso-osmotic Ringer solution after hyperosmotic exposure.
Grading of conduction velocity changes with extracellular osmolarity
Figure 2 summarises typical traces of action potentials (column A) and dV/dt (column B) for experiments that systematically investigated the effects of graded changes in extracellular osmolarity reflecting different extracellular sucrose concentrations on conduction velocity, action potential waveform, and dV/dt obtained at 7°C. Action potential waveforms again included both early rapid surface action potential deflections following the stimulus artefact and prolonged after-depolarisation phases that lasted well beyond 20 ms, reflecting T-tubular activation, at all the extracellular osmolarities studied (cf. Adrian and Peachey 1973). Measurements of latencies between the stimulus artefacts to the maximum value of the dV/dt traces then suggested a noticeable overall decrease in the calculated conduction velocities with increasing osmolarity. All these changes occurred in the absence of any excessive depolarization in resting membrane potential over the range of explored osmolarities. Thus resting potential at an osmolarity of 250 mOsm was −85.27 ± 1.05 mV (n = 87); that at an osmolarity of 700 mOsm was −75.94 ± 2.20 mV (n = 33). However, resting potential at an osmolarity of 750 mOsm was −62.71 ± 2.82 mV (n = 17) (cf. Fraser et al. 2006). At the latter extracellular osmolarity a small proportion of action potentials appeared to display markedly reduced amplitudes resulting in peak deflections that failed to show overshoots, and a firing of two consecutive action potentials after a single stimulation in the case of one fibre. Accordingly, the investigations were not performed at higher sucrose concentrations.Fig. 2Typical action potential (A) and dV/dt (B) traces recorded in the following extracellular osmolarities: (a) 250, (b) 400, (c) 500, (d) 600, (e) 650 and (f) 750 (all values given in mOsm). There is a notable overall increase in the latencies measured from (B)
Plots of conduction velocity and maximum dV/dt with increasing extracellular osmolarity
Figure 3 summarizes the results of systematic studies of the dependence of conduction velocity on extracellular osmolarity in a statistically larger number of muscle fibres. Action potentials and their corresponding dV/dt were measured systematically in n = 87, 18, 27, 11, 10, 33 and 13 fibres at sucrose concentrations of 0, 150, 250, 350, 400, 450 and 500 mM corresponding in turn to osmolarities of 250, 400, 500, 600, 650, 700 and 750 mOsm respectively. Figure 3 demonstrates that both conduction velocity (a) and maximum dV/dt (means ± standard errors of the mean) (b) monotonically decreased with extracellular osmolarity. Figure 3a then compares these experimental values of conduction velocity against predictions from the two hypotheses for the dependence of Ri on osmolarity. In both cases, θ was calculated from Ri and the extracellular osmolarity, osm, using the relationship derived from Eqs. (4) and (5) above that:Fig. 3Experimentally measured conduction velocities (a) and maximum dV/dt (mean ± standard error of the mean) plotted against extracellular osmolarity (◆). Dashed lines in (a): velocities predicted for a situation in which Ri decreases proportionally as a result of increased intracellular ionic strength produced by changes in cell volume brought about by the osmolarity changes. Continuous line: predictions when Ri increases with increasing extracellular osmolarity suggested by Hodgkin and Nakajima (1972). The changes in (a) were accompanied by decreases in maximum dV/dt (b) with increasing osmolarity, as expected for propagation brought about by local circuit currents
The dotted line denotes expectations from the first situation outlined above in which Ri decreases proportionally with increasing extracellular osmolarity: this predicts that conduction velocity does not alter with osmolarity from its control value in iso-osmotic solutions corresponding to a constant value of 0.85 m s−1 obtained in iso-osmotic solutions. The continuous line represents the second hypothesis in which Ri depends both on temperature and extracellular osmolarity as reported by Hodgkin and Nakajima (1972). The function displayed assumes the sarcoplasmic conductivity Gi(= 1/Ri) to have a Q10 = 1.37, and a value of Ri at 2°C, of 298.51 Ω cm in a muscle fibre within an extracellular solution of 250 mOsm osmolarity and of 390.63 Ω cm in a muscle fibre within an extracellular solution of 600 mOsm osmolarity, as suggested by Hodgkin and Nakajima (1970, 1972). Then, let θ1, Ri1 and osm1 correspond to conditions in iso-osmotic Ringer and θ2, Ri2 and osm2 correspond to conditions in any hyperosmotic solution. Thus:Since θ1 = 0.85 ± 0.029 m s−1 in iso-osmotic solution,where
The values generated by the above equations predicted a decline in conduction velocity with increasing extracellular osmolarity.
To test the statistical significance of the goodness-of-fit of the two contrasting predictions to the experimental data obtained, an Fx test that takes into account the difference of the two reduced-χ2 values in proportion to the first χ2 term was performed (Bevington 1969). This derived for each hypothesis a value of χ2 depicting the summed squared deviation of the original data, yi, to the predicted findings, y(xi), obtained at any extracellular osmolarity, xi, such that:
Values for χ2 obtained in the two cases, χ12 where the predicted conduction velocity is a constant value and χ22 where there is an increase in Ri with extracellular osmolarity were 21.360 and 15.141 respectively. These values were then used to compute a F-statistic, given by:
where n, the sample size = 200 and n−1 is the number of degrees of freedom.
The reduced-χ2 tests for goodness-of-fit yielded a F-statistic of 81.73 consistent with a significantly better fit (P << 0.001) to predictions from a situation in which Ri increased as described by Hodgkin and Nakajima (1972) rather than a decrease in Ri with increasing extracellular osmolarity.
Discussion
This paper begins from classic analyses (Hodgkin 1954; Adrian 1975) of the dependence of action potential conduction velocities upon the diameter of nerve fibres studied in large volume conductors under iso-osmotic extracellular conditions. This had employed cable analysis that attributed these propagation processes to local circuit currents driven by Na+ currents, INa, generating the rising phase of the action potential that in turn discharged initially passive circuit components (Valdiosera et al. 1974) equivalent to a membrane capacitance per unit area, Cm and resistance Rm of unit membrane area in series with an axial cytoplasmic resistance per unit length, ri. The latter is related to fibre radius a and sarcoplasmic resistivity Ri (kΩ cm) by Ri = riπa2 giving the original result that conduction velocity would vary as the square root of a.
The present paper extends this analysis to the effects of varying extracellular osmolarity on conduction velocities of surface membrane action potentials in Rana esculenta skeletal muscle, a situation that differed in a number of respects. First, skeletal muscle contains excitable transverse (T-) tubular membrane system responsible for initiating excitation-contraction coupling (Adrian et al. 1969, 1970; Huang 1993; Nielsen et al. 2004; Stephenson 2006). Nevertheless, the rapid initial sarcolemmal membrane activation that ensures rapid action potential propagation producing a synchronous sarcomeric activation likely largely precedes full tubular excitation (Adrian and Peachey 1973). Recent detubulation experiments left surface membrane conduction velocities unchanged suggesting a separation of surface and T-tubule excitation, the latter possibly initiated separately by Na+ channels localized around the T-tubular luminal mouths (Sheikh et al. 2001). Increasing extracellular osmolarity did not increase the diameter of the necks of the T-tubules important in electrical connectivity between T-tubule network and the fibre membrane (Launikonis and Stephenson 2002, 2004). These findings would permit muscle fibre conduction velocities to be analysed in terms of surface cylinders. Second, skeletal muscle volume alters inversely with extracellular osmolarity (Blinks 1965; Ferenczi et al. 2004) in turn correspondingly increasing solute concentration, in contrast to the situation represented by comparisons of nerve fibres of different diameters in similarly iso-osmotic extracellular solutions.
This paper then derived quantitative consequences from two possible hypotheses emerging from the above conditions. On the one hand, classical electrolyte theory (Robinson and Stokes 1959; Atkins 1998) predicts a specific conductance Ksp attributable to each intracellular ion species increasing proportionally with solute concentration following decreases in cell volume in hyperosmotic solution. This led to a prediction of a decrease in Ri precisely correcting out effects of any osmotically induced diameter change together leaving conduction velocity constant. On the other hand, empirical observations suggesting increases in Ri with extracellular osmolarity (Hodgkin and Nakajima 1972) permitted construction of a quantitative formula for the resulting variations in Ri with extracellular osmolarity as well as temperature. This led to predictions of a conduction velocity that decreased with increasing extracellular osmolarity.
The experiments described in this paper then sought to distinguish between the two hypotheses. It investigated the effects of extracellular osmolarity on conduction velocities of surface membrane action potentials in surface fibres, that would be maximally exposed to these solution changes, in frog skeletal muscle, following stimulation at a Vaseline seal at defined distances from the microelectrode recording site. Simultaneous records were made of the rate of change of membrane potential dV/dt that would be provided a consistent time point at which there would be a maximal action potential slope as well as rate of discharge of the membrane capacitance, Cm, by local currents driven by the Na+ current INa.
These studies showed that conduction velocity declined with increasing extracellular osmolarity along with maximum dV/dt as expected for a process dependent upon a local circuit current flow, despite relatively constant resting membrane potentials. These changes in conduction velocity in hyperosmotic solution were at least partially reversible. Furthermore, there were no changes in the nature of the action potential waveforms including early surface deflections and late after-depolarisation phases observed, consistent with a minimal change in the capacity for T-tubular excitation, through a still intact tubular system, that nevertheless followed generation of the initial surface component of the action potential wave.
Further systematic study in larger fibre numbers through a range of osmolarities (250–750 mOsm) demonstrated corresponding declines in conduction velocity and maximum dV/dt. At the highest osmolarity (750 mOsm), a small proportion of fibres variously showed low action potential overshoots which however did not correlate with the situations where there was a reduced conduction velocity, as well as multiple firing in a single muscle fibre. Nevertheless, further studies were not made at these and higher osmolarities; in any case, muscle fibres are thought only to act close to perfect osmometers at osmolarities up to around four times that of the iso-osmotic solution (Blinks 1965).
These findings are thus consistent with the second hypothesis in which sarcoplasmic resistivity, Ri, increases with the fibre shrinkage observed in hyperosmotic solutions, as suggested by Hodgkin and Nakajima (1972), as opposed to the first possibility outlined above in which conduction velocity should be constant. This was borne out by objective statistical analysis of the goodness-of-fit of the predictions derived from the two contrasting hypotheses as expressed in their resulting reduced-χ2 values, with the experimental values of conduction velocity generally assuming slightly higher values than predicted. The latter might reflect minor departures from a purely continuous conduction as postulated for unmyelinated cylindrical fibres, either due to contributions from peripheral regions of tubular membrane less isolated than the remaining tubular system (Hodgkin and Nakajima 1972), or the clustering of sodium channels around the tubular necks as reported by Sheikh et al. (2001) that may contribute to a more saltatory-like conduction in the muscle that would be expected to generally speed up propagation of electrical activity.
Electronic supplementary material
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Pediatr_Nephrol-3-1-1794138 | Pathophysiology of focal segmental glomerulosclerosis
| Focal segmental glomerulosclerosis (FSGS) is a major cause of idiopathic steroid-resistant nephrotic syndrome (SRNS) and end-stage kidney disease (ESKD). In recent years, animal models and studies of familial forms of nephrotic syndrome helped elucidate some mechanisms of podocyte injury and disease progression in FSGS. This article reviews some of the experimental and clinical data on the pathophysiology of FSGS.
Learning objectives Discuss the experimental and clinical data on the pathophysiology of FSGSReview the alterations in glomerular structure and function associated with FSGSTo identify potential mechanisms responsible for disease progression in FSGSDistinguish some targets for the future therapy of FSGS
Focal segmental glomerulosclerosis (FSGS) is a disease entity defined by findings on kidney biopsy [1, 2]. FSGS is the major cause of idiopathic steroid-resistant nephrotic syndrome (SRNS) in children and adults [3]. FSGS is the most common cause of acquired chronic renal insufficiency in children and frequently leads to progression to end-stage kidney disease (ESKD) [2]. FSGS may occur secondary to such disparate disease processes as HIV and obesity [1, 4]; this review focuses on the pathophysiology of primary FSGS (i.e., with no underlying illness).
Alterations of normal glomerular structure and function have been found in FSGS [5]. Normal function requires that the three major components of the glomerular filter (the endothelial cells, podocytes, and glomerular basement membrane) are intact and are able to provide a permselective filtration barrier (Fig. 1). Specialized tight junctions between podocyte foot processes create the slit diaphragm which is integral to preventing the loss of protein into the urinary space [6]. While the clinical presentation of FSGS is often heterogeneous, a characteristic feature of the disease is proteinuria, which implies the loss of this barrier [2, 7]. Indeed, electron microscopy has shown distortion of the normal architecture (or effacement) of the foot processes of podocytes in FSGS [1]. The connection between these projections of the epithelial cell and the underlying basement membrane can be disrupted, leading to the loss of nonspecific plasma proteins into the tubular filtrate [6].
Fig. 1A Components of the normal glomerular filtration barrier: (1) glomerular basement membrane (GBM); (2) podocyte foot process; (3) endothelial cell; B Progressive changes seen in focal segmental glomerulosclerosis (FSGS) leading to sclerosis: (1) foot process effacement; (2) podocyte apoptosis/loss and exposed glomerular basement membrane; (3) filtration of non-specific plasma proteins; (4) capillary expansion; (5) formation of synechiae; (6) misdirected filtration at points of synechiae; (7) mesangial matrix proliferation. Adapted from Kwoh et al. [9]
However, unlike other causes of proteinuria and nephrotic syndrome, such as minimal change disease (MCD), FSGS often progresses to ESKD. While foot process effacement is seen in MCD as well as FSGS, histologically, FSGS is characterized by increased extracellular matrix within the glomerular tuft with obliteration of the glomerular capillary lumen. These sclerotic lesions occur focally and in only some segments of glomeruli, and are typically not associated with immune complex deposition [1]. The location of sclerotic lesions by light microscopy defines the variants of FSGS: perihilar variant (with sclerosis of the vascular pole), cellular variant (associated with hypercellularity of the capillary space), tip variant (involving the part of the glomerulus near the origin of the proximal tubule), and collapsing variant (with one or more glomeruli with global or segmental collapse) [1]. Clinically, the variants of FSGS differ; for example, the collapsing variant tends to progress more rapidly to ESKD and commonly occurs in the setting of HIV [1]. It is possible that different mechanisms may play a role in the pathogenesis of each variant of FSGS [7, 8].
Insight into the pathogenesis of FSGS developed over the past decade from studies of genetic mutations in mice, models of progressive glomerulosclerosis (such as the rat remnant kidney model), and identification of gene mutations in some familial forms of nephrotic syndrome (including congenital nephrotic syndrome and familial and autosomal dominant FSGS) [7, 9, 10].
Key in the pathogenesis of FSGS is podocyte damage and loss [5, 6]. Injury to podocytes occurs by four major mechanisms: alteration of the components of the slit diaphragm or interference with its structure, dysregulation of the actin cytoskeleton, alteration of the glomerular basement membrane or its interactions with the podocyte, or alteration of the negative surface charge of the podocyte [6, 9]. Damage to podocytes triggers apoptosis and their detachment of podocytes from the glomerular basement membrane [6, 9]. The ensuing reduction in podocyte number is felt to play an important role in the pathogenesis of FSGS [7]. The podocyte is normally a terminally differentiated cell with limited proliferative capacity in response to injury [7]. The initial insult to the podocyte leads to further damage mediated by cytokine release, mechanical stress, and further loss of polarity, resulting in sclerosis and scarring of the glomerulus [7, 9].
Genetic mutations seen in congenital forms of nephrotic syndrome and FSGS enabled researchers to identify specific gene mutations involved in podocyte damage [10]. Mutations of the nephrin gene, a podocyte-specific transmembrane component of the slit diaphragm, are found in congenital Finnish-type nephrotic syndrome, and may lead to loss of normal caliber slit diaphragms [6, 9–11]. In mouse models, mutations of nephrin-like transmembrane genes (NEPH-1) which also localize to the slit diaphragm result in proteinuria and early death [6, 10].
It is unclear how alteration of the slit diaphragm results in podocyte loss. The slit diaphragm may be integral to maintaining cell polarity or its damage may alter the balance of cell signals, resulting in apoptosis. Mutations in a Fyn kinase (one of the src tyrosine kinases) that phosphorylates nephrin and may regulate cell cycle and apoptosis resulted in proteinuria and foot process effacement in a mouse model [9, 10].
Other proteins which are part of the slit diaphragm complex include: podocin, CD2-associated protein (CD2AP), FAT, ZO-1, P-cadherin, an LAP (leucine rich repeat and PDZ domain) protein, and MAGI-1 [6, 10]. Mutations in podocin (a transmembrane protein that interacts with nephrin, NEPH-1 and CD2AP) have been identified in familial FSGS [9, 10, 12]. Recently, mutations in CD2AP, an immunoglobulin-like protein that is involved in nephrin integration with the podocyte cytoskeleton, have also been linked to genetic forms of FSGS [10, 13, 14]. In mouse models, the loss of FAT1 and FAT2 (transmembrane proteins with cadherin-like repeats) results in the absence of slit diaphragms, proteinuria, and early death [10]. The role of the other components of the slit diaphragm in the pathophysiology of FSGS is not yet clear.
Alpha-actinin-4, an important structural component of the podocyte cytoskeleton, is mutated in some autosomal dominant forms of FSGS [10, 15–17]. Other mutations have been identified in association with FSGS in addition to abnormal structural proteins. For example, TRPC6 is a cation-selective ion-channel protein that mediates calcium signals and has also been associated with FSGS [18].
Certain clinical variants of FSGS are suggestive of different mechanisms of injury to the podocyte. For example, a circulating factor which leads to glomerular basement membrane injury has been proposed in the pathogenesis of some types of FSGS [19, 20]. For example, there appears to be a role of a circulatory factor in the recurrence of FSGS in transplanted kidneys [20]. In some patients with recurrent FSGS, proteinuria remits in response to plasmapheresis and the removal of serum proteins. In addition, injections of serum from patients with recurrent FSGS were capable of inducing proteinuria in rats [20].
Another example of alternative mechanisms of injury is collapsing FSGS, which occurs in the setting of viruses such as HIV. In collapsing FSGS, dysregulation of the podocyte cell cycle appears to result in immature, proliferative podocytes [21, 22]. Finally, recent work has focused on the role of the parietal epithelial cell in the pathophysiology of FSGS [23]. Proliferation of parietal epithelial cells was identified in both a transgenic model of FSGS and a biopsy from a patient with collapsing FSGS [23].
Of great clinical importance is the mechanism by which the initial podocyte injury progresses to the final sclerotic lesion (Fig. 1). As podocyte numbers decline, there is a relative exposure of the glomerular basement membrane. Maladaptive interactions develop between the glomerular basement membrane and the parietal epithelial cells. Expansion of synechiae and/or the leak of protein into Bowman’s space results in the deposition of collagen. Ultimately, this results in the collapse of the capillary loop and the loss of endothelial cells [5].
Factors resulting in the progression of FSGS to ESKD have also been the focus of recent research (Fig. 2). Cytokines and vasoactive factors are believed to play a major role in the progression of FSGS. The overexpression of transforming growth factor β (TGFβ) or its effector proteins, the Smads, leads to glomerulosclerosis in animal models [8, 24]. Activation of the renin-angiotensin system upregulates TGFβ and is felt to further lead to the progression of disease [7, 24]. Other angiogenic factors, such as platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) may also play a role in disease progression [24]. The evidence for this is primarily based on animal models of progressive glomerulosclerosis, such as the rat remnant kidney model. In this model, PDGF and VEGF are upregulated and the later loss of VEGF expression correlates with progression of the glomerulosclerosis [24, 25].
Fig. 2Factors involved in the progression of FSGS to end-stage kidney disease (ESKD): initial loss or injury to podocytes (related to defects in membrane proteins or cytoskeleton instability) leads to cytokine release, mechanical stress, hyperfiltration, and glomerular hypertrophy. These factors lead to upregulation of an inflammatory response mediated by monocytes, macrophages, and T-cells. The end result is collagen matrix deposition and fibrosis, and progression to ESKD
Mechanical stress is also believed to play a role in the progression of FSGS [9, 26]. Increased filtration due the defects of the filtration barrier results in increased single-nephron glomerular filtration rate (SNGFR). This hyperfiltration results in hypertrophy of glomeruli. The hypertrophy exacerbates the mismatch between the glomerular basement membrane and the decreased numbers of podocytes, resulting in further injury [9].
Another factor in the progression of FSGS is tubulointerstitial injury. Clinically, tubulointerstitial injury is a predictor of the loss of renal function in FSGS [1, 27]. The nonspecific entry of proteins into the tubular lumen is one potential source of damage to the interstitium. Indeed, persistence of nephrotic-range proteinuria is a negative prognostic factor for the progression of FSGS to ESKD [28]. While it is unclear if proteinuria itself is toxic to the tubulointerstitium, decreases in proteinuria achieved by angiotensin-converting enzyme (ACE) inhibitors and by angiotensin receptor blockers (ARB) appear to slow disease progression in some adults with FSGS [9, 29].
The presence of plasma proteins in the tubular filtrate may directly injure the tubulointerstitium. Cytokines (such as TGFβ), when present in the tubules, will recruit monocytes, macrophage, and T-cells. This stimulates other cytokines, including interleukin-1, tumor necrosis factor alpha, and other chemokines [24]. The inflammatory infiltrate leads to mesangial matrix deposition, promoting the collapse of glomeruli. The cellular infiltrate and cytokines also damage tubular epithelial cells, and some tubular epithelial cells may undergo transformation to mesenchymal cells (an epithelial-mesenchymal transition or EMT) [24]. These mesenchymal cells, as well as recruited and stimulated fibroblasts, result in collagen matrix deposition and tubulointerstitial fibrosis [24].
The beneficial effects of blocking the renin-angiotensin system may not be limited to their antiproteinuric or antihypertensive effects. As noted earlier, angiotensin stimulates TGFβ, contributing to fibrosis. It can also induce oxidative stress and it is stimulated by mechanical stress, such as hyperfiltration [24]. In addition, angiotensin affects intracellular calcium concentrations and the podocyte cytoskeleton [24]. Inhibition of angiotensin may slow progression by these local mechanisms [9, 29].
With the increasing incidence of FSGS in children [30], these pathways of podocyte injury and disease progression provide important targets for future intervention. Trials have already been initiated to antagonize cytokines, such as TGFβ. Future therapeutic targets may include factors involved in podocyte protection or tubulointerstitial injury.
Questions (Answers appear following the reference list)
Which of the following statements is TRUE regarding the current understanding of the pathogenesis of focal segmental glomerulosclerosis (FSGS)?
FSGS may result from immune-complex-mediated damage to endothelial cellsAlterations in components of the slit diaphragm may play a role in the pathogenesis of FSGSProliferation of podocytes leads to cytokine release and mechanical stress, resulting in scarring and sclerosis of the glomeruliMutations in a chloride channel have been associated with FSGS and may be pathogenicAll of the following are mutations of structural proteins that have been identified as pathogenic in FSGS EXCEPT:
Sodium channel mutationAlpha-actinin-4NephrinPodocinProgression of FSGS to end-stage kidney disease (ESKD) results from:
Downregulation of transforming growth factor β (TGFβ)Decreased glomerular filtrationTubulointerstitial injuryBlockade of the renin-angiotensin systemProteinuria in the setting of FSGS:
Has no effect on clinical courseMay be decreased by treatment with angiotensin-converting enzyme (ACE) inhibitorsResults from an increased number of glomerular foot processesLeads to the loss of mesangial matrixWhich of the following is FALSE:
A circulating factor may play a role in the pathogenesis of FSGSProliferation of parietal epithelial cells has been identified in collapsing FSGSPodocyte loss due to necrosis appears to play a role in the pathogenesis of FSGSCD2-associated protein, FAT, nephrin, and podocin are examples of slit diaphragm proteins | [
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"animal models",
"podocyte",
"injury",
"nephrin",
"podocin",
"tubulointerstitial",
"transforming growth factor (tgfβ)"
] | [
"P",
"P",
"P",
"P",
"P",
"P",
"P",
"P",
"P",
"R"
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Neurosurg_Rev-2-2-1564192 | Clinical and radiological features related to the growth potential of meningioma
| Clinical and radiological features that help predict the growth potential of meningioma would be beneficial. The purpose of this study is to clarify the characteristics related to proliferating potential using the MIB-1 staining index. We analyzed the relationship of MIB-1 staining indices to characteristics of 342 consecutive patients with meningioma surgically removed between 1995 and 2004 by logistic regression analysis. One hundred and forty-nine of the patients with meningioma were ≥60 in age; 89 male; 48 recurrent; 203 symptomatic; 157 at the skull base; 124 over 20 cm3; 24 multiple; 136 with edema; 117 with calcification. The MIB-1 staining index in 56 of 296 grade I meningiomas in WHO classification was ≥ 3.0; in 27 of 28 grade II; and in 17 of 18 grade III, respectively. Logistic regression analysis demonstrated that male (odds ratio [OR], 2.374, p=0.003), recurrence (OR, 7.574, p=0.0001), skull base (OR, 0.540, p=0.035), calcification (OR, 0.498, p=0.019) were independent risk factors for a high MIB-1 staining index (≥3.0); age, symptomatic, volume, multiple, edema were not. Male, recurrence, non-skull base, absence of calcification are independent risk factors for a high proliferative potential. These should be taken into consideration when managing meningiomas.
Introduction
An estimated 2–3% of the population has an incidental asymptomatic meningioma in autopsy studies [14, 20]. With the wider use of CT and MRI, many meningiomas are discovered as incidental findings during investigation for unrelated symptoms [9, 13, 16, 17, 20, 21]. The growth potential of meningiomas varies. Some meningiomas remain unchanged in size for a long period, whereas others grow rapidly [15]. Sex, age, initial tumor size, and calcification were reported to be related to the tumor growth judging from follow-up scans [9, 13, 16, 17, 21].
The nuclear antigen Ki-67 expressed by proliferating cells has become available for routinely processed paraffin section. The MIB-1 antibody detects an epitope on the Ki-67 antigen, a nuclear protein present only during active phase of the cell cycle (G1, S, G2, and M) [2]. Several studies investigated how Ki-67 labelling indices could help to predict recurrence [1, 3, 4, 6–8, 11, 12, 18, 19]. An increased MIB-1 staining index was highly correlated with a shorter tumor volume doubling time [12]. In the cases that showed an MIB-1 of ≥ 3%, the tumor volume doubling time was <2 years. Nakaguchi et al. [12] found the formula which can calculate tumor doubling time (Td) from the MIB-1 staining index at surgery: log Td=31.4–0.14×MIB-1 SI (R2=0.556). The time interval to the next recurrence for recurrent meningiomas is associated with the MIB-1 staining index. Meningiomas with MIB-1 staining index of 3% or higher had a significantly higher tendency of recurrence [11]. Although these cell kinetics methods are valuable for growth potential, they can be applied only after the verification of pathology [15].
The purpose of this study was to clarify the clinical and radiological features related to meningioma proliferation using the MIB-1 staining index. Sex, age, calcification, edema, symptom, size, and shape of tumor were already reported to be related to the tumor growth and MIB-1 staining index [1, 3, 4–8, 11–13, 16–19, 21]. The results, however, were inconsistent because of a lack of sufficient case number and inadequate statistical analysis. Independent risk factors for high growth rate should be determined. We analyzed the relationship of MIB-1 staining indices to the characteristics of 342 consecutive patients with meningioma surgically removed between 1995 and 2004 by logistic regression analysis.
Materials and methods
Three hundred and forty-two patients with meningiomas were surgically treated in our department of neurosurgery between 1995 and 2004. Radiological features were analyzed by CT scans and/or MRI. Location of the tumor was classified as follows: convexity, falx, parasagittal, sylvian fissure, tentorial, ventricular, foramen magnum, olfactory groove, petroclival, petrous, sphenoid ridge, and tuberculum sellae. The latter six locations were considered as skull base. The tumor volume was calculated using the formula: length × depth × width × 0.5 [9]. When patients had multiple meningiomas, only the largest tumor was included. On the basis of conventional CT and bone window CT, patients were divided into two groups according to the low density area around the tumor and calcification in the tumor. A low density area due to surgical scar was not included in edema in patients with recurrent meningioma.
The tumors were histologically classified according to the World Health Organization classification of tumors [10]. An avidin-biotin immunoperoxidase or simple stain MAX-peroxidase (Nichirei, Tokyo) technique was used to perform MIB-1 monoclonal antibody (DAKO, Denmark) assay in selected sections of each case. All tissue sections were examined at high-power magnification (×400). The number of cells stained positively with MIB-1 and the total number of tumor cells were counted in several representative fields containing more than 1,000 cells. Their ratio was indicated as the MIB-1 staining index (%).
Statistical analysis
All data were stored on a personal computer and analyzed using commercially available statistical software (SPSS version 12.0, SPSS Inc.). Chi-squired analysis was used to compare the MIB-1 staining index to characteristics of patients with meningioma. All variables were included in a logistic regression model to determine which variables were independently associated with a high MIB-1 staining index (≥3.0). Significance was judged at a value of p<0.05 for all analyses.
Results
Table 1 shows the characteristics and MIB-1 staining index of the 342 patients. One hundred and forty-nine of patients with meningioma were ≥60 in age; 89 male; 48 recurrent; 203 symptomatic; 157 at the skull base; 124≥20 cm3 in volume; 24 multiple; 136 with edema; 117 with calcification. We compare these characteristics to the MIB-1 staining index. We divided them into two groups: <3.0 and ≥3.0 [11]. The MIB-1 staining index in 100 of 342 meningiomas was > 3.0. Sex (p=0.0001), recurrence (p=0.0001), symptomatic (p=0.013), volume (p=0.014), edema (p=0.001), and calcification (p=0.0001) were correlated with the MIB-1 staining index by chi-square test; age, skull base, and multiple were not.
Table 1Characteristics and MIB-1 staining index in 342 meningiomas MIB-1 staining index (%) Factor<3.0≥3.0P ValueAge (years)0.937-49652450-723260-793470-2610Sex (male/female)46/19643/570.0001Recurrence (yes/no)14/22834/660.0001Symptomatic (yes/no)133/10970/300.013Skull base (yes/no)119/12338/620.059Volume (cm3)0.014-9.91133710-531520-7648Multiple (yes/no)15/2279/910.244Edema (yes/no)82/16054/460.001Calcification (yes/no)97/14520/800.0001Total242100
Meningothelial, transitional, and fibrous meningiomas were the three major subtypes, and they accounted for about three fourth of the total. Two hundred and ninety-six meningiomas belonged to grade I; 28 grade II; and 18 grade III. MIB-1 staining index in 56 of 296 grade I meningiomas was ≥ 3.0; that in 27 of 28 grade II; and that in 17 of 18 grade III, respectively (Table 2).Table 2Histological subtypes and MIB-1 staining index of 342 meningiomasSubtypeMIB-1 staining index (%)Total<3.0≥3.0Grade IMeningothelial 10628134Fibrous671380Transitional431356Psammomatous606Angiomatous13215Microcystic101Secretory101Lymphoplasmacyte-rich101Metaplastic20224056296Grade IIAtypical12627Chordoid01112728Grade IIIRhabdoid011Papillary112Anaplastic0151511718Total242100342
Logistic regression analysis demonstrated that male (odds ratio [OR], 2.374, p=0.003), recurrence (OR, 7.574, p=0.0001), skull base (OR, 0.540, p=0.035), calcification (OR, 0.498, p=0.019) were independent risk factors for a high MIB-1 staining index (≥ 3.0); age, symptomatic, volume, multiple, and edema were not (Table 3).
Table 3Logistic regression analysis for factors independently related to MIB-1 staining indexFactorOdds ratio95%CIP ValueAge1.1090.841–1.4610.464Sex2.3741.336–4.2190.003Recurrence7.5743.558–16.1240.0001Symptomatic1.4680.774–2.7840.240Skull base0.5400.305–0.9560.035Volume1.3320.944–1.8790.103Multiple1.0270.398–2.6510.957Edema1.5080.838–2.7110.170Calcification0.4980.278–0.8920.019
Discussion
We analyzed the relationship of the MIB-1 staining indices to the characteristics of 342 consecutive patients with meningioma surgically removed between 1995 and 2004 by logistic regression analysis. Logistic regression analysis demonstrated that male (odds ratio [OR], 2.374, p=0.003), recurrence (OR, 7.574, p=0.0001), skull base (OR, 0.540, p=0.035), calcification (OR, 0.498, p=0.019) were independent risk factors for a high MIB-1 staining index (≥ 3.0); age, symptomatic, volume, multiple, and edema were not.
The relationship between the growth rate or MIB-1 staining index and age has been controversial: a higher MIB-1 staining index and higher growth rate were observed for younger patients [11, 13, 21]; but not in other reports [1, 12, 15, 19]. Our series of 342 patients with meningioma showed no relation.
It is well known that atypical and anaplastic meningiomas are predominant in males [10]. Matsuno et al. [11] reported that the mean MIB-1 staining index in 50 male patients was 5.5%, whereas that in 77 female patients was 2.7%. Our findings show that male (odds ratio [OR], 2.374, p=0.003) was an independent risk factor for a high MIB-1 staining index. We also found a higher MIB-1 staining index in males even in grade I meningioma (MIB-1 staining index in 32 of 226 females, and in 24 of 70 males was ≥ 3.0, p=0.0001, chi-squire test).
Recurrence (OR, 7.574, p=0.0001) was the most significant independent risk factor for a high MIB-1 staining index (≥ 3.0). Therefore, we propose prompt management for recurrent meningiomas. In most of the recurrent meningiomas, the MIB-1 staining index was higher at the time of recurrence than at the time of initial surgery [1, 11, 19]. Changes in histological morphology and malignant transformation are also known in meningiomas.
Although there is a significant difference in the MIB-1 staining index between symptomatic and non-symptomatic meningiomas by chi-square test, symptomatic is not an independent risk factor for a high MIB-1 staining index. Meningiomas commonly present with seizure disorders, and are associated with location, perilesional edema, and convexity location. Symptoms and signs of elevated intracranial pressure could be due to the large size of meningioma itself, or to the pronounced cerebral swelling resulting from reactive vasogenic edema. Focal neurological deficits caused by meningiomas are generally related to direct local brain, cranial-nerve compression, and can be predicted from the site of origin of the tumor [20]. Thus, symptomatic meningioma may not be related to a high MIB-1 staining index. The growth rate of incidental meningioma may be similar to that of symptomatic meningioma.
Our results demonstrated that skull base (OR, 0.540, p=0.035) is an independent risk factor for a high MIB-1 staining index. No relationship has been reported between the MIB-1 staining index and the location of meningiomas [15, 19]. In general, the surgical risk for meningiomas is higher in skull base. A low proliferative potential in skull base meningiomas should be taken into consideration especially when treating elderly patients with asymptomatic meningiomas [9, 16].
Although there was a significant difference of the MIB-1 staining index in tumor size by chi-square test [15], tumor size is not an independent risk factor for a high MIB-1 staining index. The tumor volume is associated with the annual growth rate but not with doubling time [13, 16, 21]. Assuming that a tumor shows a constant relative volume increase, larger tumors will show higher growth rates if the annual volume increase is expressed in absolute values. Large tumors should be carefully observed even though the initial volume is not a risk factor for a high MIB-1 index, and it is not correlated with doubling time.
The etiology of peritumoral brain edema associated with meningiomas is multifactorial. Factors that may influence the etiology of peritumoral edema include tumor size, histological subtypes, vascularity, venous stasis, and brain invasion [1, 4]. Ide et al. [4] found a significant correlation of both the MIB-1 staining index and tumor size with the extent of edema. A high MIB-1 staining index itself did not seem to be directly responsible for perifocal edema, since our logistic regression analysis demonstrated that edema is not an independent risk factor for a high MIB-1 index [15].
Tumors with calcification grew significantly less than those without calcification [9, 13, 16]. Absence of calcification on CT correlated strongly with doubling time [5]. Diffusely calcified meningiomas had a low mean MIB-1 staining index of 0.57%. Focally calcified tumors showed a relatively low proliferative potential (0.92%) compared with that of noncalcified tumors (1.75%) [15]. The results are always consistent when the relationship between calcification and proliferative potential or growth rate is compared. We also confirmed this characteristic in meningioma (OR, 0.498, p=0.019).
In conclusion, male, recurrence, non-skull base, absence of calcification are independent risk factors for a high proliferative potential. These should be taken into consideration when managing meningiomas. | [
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Mech_Dev-2-1-2428104 | Polydactyly in the mouse mutant Doublefoot involves altered Gli3 processing and is caused by a large deletion in cis to Indian hedgehog
| The mouse mutant Doublefoot (Dbf) shows preaxial polydactyly with 6–9 triphalangeal digits in all four limbs and additional abnormalities including a broadened skull, hydrocephalus, and a thickened, kinked tail. The autopod undergoes a characteristic expansion between late embryonic day (E) 10.5 and E11.5, following the onset of ectopic Indian hedgehog (Ihh) expression in the entire distal mesenchyme, except for the zone of polarising activity (ZPA), at E10.5. We show here that limb prepattern, as indicated by expression of Gli3 and Hand2 at E9.5 is unaffected by the mutation. As both Sonic hedgehog (Shh) and Ihh expression are present in Dbf limb buds at E10.5, we generated Dbf/+;Shh−/− mutants to analyse the effects of different patterns of Hedgehog activity on the limb phenotype and molecular differentiation. Dbf/+ embryos lacking Shh showed postaxial as well as preaxial polydactyly, and the Ihh expression domain extended posteriorly into the domain in which Shh is normally expressed, indicating loss of ZPA identity. Differences in gene expression patterns in wild type, single and compound mutants were associated with differences in Gli3 processing: an increased ratio of Gli3 activator to Gli3 repressor was observed in the anterior half of Dbf/+ limb buds and in both anterior and posterior halves of compound mutant limb buds at E10.5. To identify the cause of Ihh misregulation in Dbf/+ mutants, we sequenced ∼20 kb of genomic DNA around Ihh but found no pathogenic changes. However, Southern blot analysis revealed a ∼600 kb deletion disrupting or deleting 25 transcripts, starting 50 kb 5′ of Ihh and extending away from the gene. The large deletion interval may explain the wide range of abnormalities in Dbf/+ mutants. However, we did not detect anologous deletions in cases of Laurin–Sandrow syndrome, a human disorder that shows phenotypic similarities to Dbf.
1
Introduction
The Dbf mutant, which arose spontaneously in the 3H1 (C3H/HeH × 101/H F1 hybrid) genetic background at Harwell (UK), is a polydactylous mouse that exhibits semidominant inheritance. Mice heterozygous or homozygous for Dbf have 6–9 digits in all four limbs; the extra digits are all triphalangeal and arise preaxially (Lyon et al., 1996; Hayes et al., 1998a). Dbf/+ mice also show malformation of the tibia, a broadened skull, hydrocephalus, a thickened kinked tail, and reduced fertility and viability. Homozygotes additionally exhibit a midline facial cleft but cannot be recovered alive beyond embryonic day (E) 14.5.
Polydactyly has been described in many mouse mutants, all except two of which show a discrete anterior domain of Sonic hedgehog (Shh) expression (Masuya et al., 1995; Hill et al., 2003). The Extra-toes (XtJ) mutant has an extended Shh domain due to functional inactivation of Gli3 (Hui and Joyner, 1993), whereas Dbf mice exhibit ectopic Indian hedgehog (Ihh) expression in the distal limb bud mesenchyme (Yang et al., 1998). Ectopic Ihh upregulation is first detectable at E10.5 (Crick et al., 2003), the stage at which hyperexpansion of the autopod begins; downstream targets of Shh signalling are ectopically up-regulated (Hayes et al., 1998b; Yang et al., 1998). However, the molecular mechanism by which the polydactyly arises from ectopic Ihh expression has not been investigated.
The polydactylous phenotype of the XtJ mutant was originally thought to result from the enlarged Shh expression domain (Hui and Joyner, 1993). However, Shh−/−;Gli3−/− mutants exhibit polydactyly in a similar pattern to Gli3−/− mutants, suggesting that the polydactyly of Gli3-deficient mice is independent of Shh (te Welscher et al., 2002). In wild type (wt) limb buds, digital number and identity are regulated by interaction between Shh and Gli3 (Litingtung et al., 2002). In the presence of Shh, Gli3 remains as a 190 kDa activator species, Gli3A, that up-regulates Hedgehog (Hh)-responsive gene expression, while in the absence of Shh, Gli3A is processed to a smaller 83–86 kDa repressor form, Gli3R, which negatively regulates expression of Shh and its target genes (Dai et al., 1999; Shin et al., 1999; Sasaki et al., 1999). Litingtung et al. (2002) suggested that in wt limb buds the Gli3A:Gli3R ratio controlled by Shh limits the polydactylous potential of the autopod, imposing pentadactyl constraint. This is supported by the localization of Shh protein in wt limb buds, which extends anterior to the zone of polarising activity (ZPA) in a domain coincident with Patched1 (Ptc1) expression (Gritli-Linde et al., 2001), resulting in a posterior-to-anterior increase of the Gli3A:Gli3R ratio (Wang et al., 2000). Consistent with these observations, the Gli3 present throughout Shh−/− limb buds is mainly processed to Gli3R (Litingtung et al., 2002). Recently, the mutation underlying the polydactylous chicken talpid3 mutant has been reported to be in a novel gene and has also been shown to result in abnormal Gli3 processing (Davey et al., 2006). Given the evidence of involvement of abnormal Gli3 processing in the XtJ, Shh−/− and talpid3 mutants, it is possible that the polydactyly present in Dbf mice also results from aberrant Gli3 processing. This hypothesis is supported by evidence that Gli3 acts downstream of Ihh during endochondral skeletal development (Hilton et al., 2005; Koziel et al., 2005).
To investigate the mechanism by which polydactyly arises in Dbf we have analysed gene expression in Dbf/+ limbs, where there is an excess of Hedgehog (Hh) signalling, and compared this to Shh−/− limbs, where there is none. Since Shh and Dbf are located on different chromosomes (5 and 1, respectively) (Blake et al., 2003; Hayes et al., 2001), we have been able to generate mutant mice that carry two copies of the disrupted Shh allele and are heterozygous for the Dbf mutation. To further dissect the mechanisms underlying the limb malformations in both Shh and Dbf mutants, we have analysed the effects of the ectopic Ihh expression associated with Dbf limb abnormalities in the Shh-null background by correlating altered patterns of gene expression with the phenotype of single and double mutants. Differences in Gli3 processing between each genotype suggest that Hh-Gli3 interactions govern the observed differences in digital number, and that postaxial polydactyly results from expression of Ihh, but not Shh, in the posterior ZPA mesenchyme.
Previous attempts to identify the Dbf mutation have been unsuccessful. Hayes et al. (2001) constructed a high resolution genetic map and localized the mutation to a 0.4 cM interval on mouse chromosome 1. This region contained 35 genes including several plausible candidates for the Dbf mutation. However, despite the sequencing of three of these genes, the Dbf mutation remained unidentified. Based on the misregulation of Ihh expression in Dbf, we sequenced ∼20 kb of the surrounding genome but found no obvious pathogenic changes. To investigate whether a genomic rearrangement could be responsible, we used the mouse genome sequence to design a Southern blotting strategy to systematically screen the regions 5′ and 3′ of Ihh for copy number changes. We identified a ∼600 kb deletion starting ∼50 kb 5′ of Ihh, which removes or interrupts 25 known and predicted transcripts. This raises the possibility that additional abnormalities seen in Dbf/Dbf mice arise from loss of function of deleted genes, in addition to Ihh misregulation.
2
Results
2.1
The prepattern of Dbf limb buds is unaffected
Expression of Hand2 and Gli3 has been implicated in patterning the limb bud prior to Shh expression, and has been shown to be affected later by the absence of Shh (Chiang et al., 2001; te Welscher et al., 2002). We assayed expression of these two genes before (E9.5) and after (E11.5) the onset of ectopic Ihh at E10.5 in Dbf/+ mutant embryos (Fig. 1). Gli3 expression is restricted to the anterior portion of the limb bud in wt embryos at E9.5 (Fig. 1A) and this expression pattern is not altered in the limb buds of Dbf/+ mutants (Fig. 1B). Hand2 is expressed throughout the flank of wt embryos prior to formation of the limb bud, then becomes limited to the posterior region of the limb bud as it is initiated (Fig. 1C); this pattern is not altered in Dbf/+ embryos at E9.5 (Fig. 1D). At E11.5, expression of Gli3 in Dbf/+ limb buds differs from that in wt embryos in extending more distally; the domain is also broader although this probably simply reflects the greater breadth of the limb bud (Fig. 1F). Expression of Hand2 is limited to the proximal posterior margin in wt E11.5 limb buds (Fig. 1G); in contrast, the Hand2 domain in Dbf/+ limb buds extends anteriorly and distally (Fig. 1H). Hence the limb prepattern as indicated by the expression of Hand2 and Gli3 at E9.5 is unaffected in Dbf/+ limb buds, but the expression domains of both genes are altered in association with the presence of ectopic Ihh expression at E11.5 (Fig. 3H).
2.2
Altered limb phenotype of Dbf mutants in the absence of Shh
As Shh-null embryos die perinatally, gross morphological examination of wt, Shh−/−, Dbf/+ and Shh−/−;Dbf/+ embryos was conducted at E13.5 and alcian blue staining of the limb bones was carried out at E17.5 (Fig. 2). Both forelimb and hindlimb autopods of Shh−/−;Dbf/+ embryos resemble those of Dbf/+ except that the broadened digital plate is more regular, shows fewer bifurcations, and is more extensive posteriorly (compare Fig. 2B, F and J with D, H and L). At E13.5 the autopod forms a 180° fan, and the angle between the autopod and zeugopod on the postaxial side of the limb is decreased to 90° (Fig. 2D, arrow).
2.3
Ihh and Shh expression in compound mutant limbs is mutually exclusive
The expansion that characterizes the Dbf/+ autopod takes place from late E10.5 to E11.5. We therefore analysed the expression domains of Shh and Ihh in limb buds immediately prior to (E10.5) and after (E11.5) the period of expansion. In both wt and Dbf/+ limb buds at E10.5, Shh is expressed at the posterior margin (Fig. 3A and B), defining this region as the ZPA (Riddle et al., 1993). In wt mice Ihh is not expressed in limbs prior to E12.5 (St-Jacques et al., 1999) while in Dbf/+ mutant mice, Ihh expression is present in the distal mesenchyme of the limb bud at E10.5 (Fig. 3C). This ectopic Ihh domain extends throughout the area anterior to the ZPA and may correspond to the progress zone. Its absence from the ZPA was confirmed by double in situ hybridization to show nonoverlapping juxtaposed Shh and Ihh expression (Fig. 3D). Expression of Ihh in E10.5 Shh−/−;Dbf/+ mutant limb buds extends throughout the distal mesenchyme including the posterior margin, i.e. the domain in which Shh is expressed in Dbf/+ embryos (Fig. 3E).
At E11.5, Shh expression continues in the posterior margin of wt and Dbf/+ limb buds (Fig. 3F and G). Expression of Ihh in Dbf/+ mutant limb buds at E11.5 is progressively down-regulated from posterior to anterior, until it remains only in the anterior margin (Fig. 3H); in contrast, in Shh−/−;Dbf/+ mutant limbs, down-regulation of Ihh expression begins mid-distally, remaining strong in both the anterior and posterior mesenchyme (Fig. 3I).
2.4
Gene expression is altered in Dbf limb buds lacking Shh
To gain insight into the mechanisms underlying the different patterns of polydactyly generated in the presence of different sources of Hh signalling in Dbf/+ and Shh−/−;Dbf/+ limbs, we examined the expression of genes implicated in Shh signalling and limb patterning in wt, Dbf/+, Shh−/− and Shh−/−;Dbf/+ limb buds at E10.5 (Fig. 4); as shown in Fig. 3, this is the stage at which Ihh expression is first detected. Expression of the transcriptional targets of Hh signalling, Ptc1 and Gli1, is expanded anteriorly in Dbf/+ limbs; interestingly, expression of these genes is broader in the proximal mesenchyme of Dbf/+limbs lacking Shh, suggesting expansion of the domain of Hh signalling in these limb buds. Conversely expression of Gli3, which is thought to be repressed by Hh signalling (Takahashi et al., 1998), shows a reduced expression domain in Dbf/+ limbs. As expected, Gli3 is expressed throughout Shh−/− limbs at E10.5, but in the presence of Ihh in Shh−/−;Dbf/+ mutants it is dramatically down-regulated and required a prolonged colour development time for detection.
In wt and Dbf/+ limbs at E10.5 there is a strong expression of Hand2 in the posterior mesenchyme, with a graded lower expression anteriorly, similar expression is seen in Shh−/− limbs. However, in Shh−/−;Dbf/+ limbs there appears to be a second strong anterior domain of Hand2 expression, consistent with the extended expression seen at E11.5 (Fig. 1H). As reported previously (Hayes et al., 1998b; Yang et al., 1998), the Hoxd13 domain is expanded anteriorly in Dbf/+ limb buds; in Shh−/−;Dbf/+ limb buds, the domain shows even greater expansion, consistent with the more regular digital fan seen in these mutants. Expression of Fgf8 throughout the AER of expanded Dbf/+ and Shh−/−;Dbf/+ limbs indicates that in both mutants Hh signalling between the mesenchyme and ectodermal AER is intact. Ectopic anterior expression of Fgf4 in the expanded limb buds of both mutants is consistent with their ectopic Ihh expression. Bmp4 expression in the progress zone was slightly down-regulated in Dbf/+ limbs but up-regulated proximally; like wt limbs, it was absent from the AER. In contrast, Shh−/−;Dbf/+ limbs, which showed further down-regulation of Bmp4 in the mesenchyme of the progress zone, showed ectopic expression throughout the AER. Explanation for this pattern requires further investigation.
2.5
The Dbf mutation affects the limb bud Gli3 ratio
The action of Gli3 protein as a transcriptional activator relies on its maintenance as Gli3A, which requires Hh signalling (Dai et al., 1999; Sasaki et al., 1999; Shin et al., 1999). To determine the effect of differential Hh signalling on Gli3 processing in mutant limb buds, we used a Gli3 antibody combined with Western blot analysis to assess the comparative levels of Gli3A and Gli3R in the anterior and posterior halves of E10.5 limb buds of all four genotypes (Fig. 5). As reported previously, wt limbs have a higher ratio of Gli3R to Gli3A anteriorly than posteriorly (Wang et al., 2000, and Fig. 5B and C). Dbf/+ limb buds have a reduced level of the repressor relative to the activator, especially in the anterior half, where levels of the two forms of Gli3 are similar. In Shh−/− limb buds the difference between the anterior and posterior halves is greatly reduced with relatively high levels of Gli3R to Gli3A in both halves of the limb bud mesenchyme (Litingtung et al., 2002, and Fig. 5B and C). In Shh−/−;Dbf/+ mutants, both halves of the limb bud show a decreased ratio of Gli3R to Gli3A compared with the wt result; the ratio is similar in both halves of the limb bud, in contrast to the Dbf/+ result which shows an A–P asymmetry.
2.6
A ∼600 kb deletion underlies Dbf
To determine the cause of the ectopic Ihh expression in Dbf limbs, we initially searched for the genetic lesion by sequencing 20 kb of the region around Ihh in Dbf heterozygotes and both parental strains, but found no pathogenic changes (data not shown). Subsequently we sought genomic rearrangements using a systematic Southern blotting strategy to interrogate the mouse genome sequence (http://genome.ucsc.edu/), which initially identified the absence of a polymorphic 8 kb SpeI fragment in Dbf (see Section 4.5). Characterization of the breakpoint by Southern analysis and subsequently by inverse PCR led to identification of the centromeric breakpoint at position 75,098,488 bp on chromosome 1 (Fig. 6). Analysis of sequence 3′ to this in Dbf/+ DNA revealed the telomeric breakpoint to be at position 75,694,480 bp on chromosome 1. The deleted region therefore appears to be 595,992 bp; however this figure is not precise because the deletion encompasses a ∼16 kb gap in the current mouse genome sequence (mm9 assembly) present between 75,102,130 and 75,118,131 bp. We confirmed the deletion by PCR using primers flanking the breakpoint and further demonstrated that three different loci distributed within the putatively deleted region were present only in a single copy in Dbf/+ mutant DNA (see Section 4.5). Analysis of the wt sequences at the two breakpoints showed that the sequence at the centromeric breakpoint is unique, lying within the gene Non-homologous end joining factor 1 (Nhej1). However, a hexanucleotide motif CCAAAC present at the breakpoint is repeated 17 nucleotides upstream, separated by four copies of a trinucleotide CCT motif. The telomeric breakpoint resides within the 3′ terminal region of a B1 repetitive element at the endpoint of a very T-rich motif (35 thymine residues in 47 bases) which is likely to represent the complement of an ancestral poly(A) tract related to the B1 element and does not disrupt any known gene. There is a three nucleotide ambiguity in the position of the breakpoint as the sequence ACA is present on both sides of the deletion (Fig. 6). In addition to disrupting Nhej1, the deletion completely removes 24 known and predicted genes (Fig. 6, Supplementary Table 2 and Section 3).
2.7
Laurin–Sandrow syndrome does not result from large deletions 5′ of IHH
Laurin–Sandrow syndrome (LSS) (MIM 135750) is rare human developmental disorder characterized by triphalangeal preaxial polydactyly of the hands and feet, with variable involvement of the proximal limb elements. It has been previously suggested that LSS shares many similarities with Dbf and may also arise from ectopic IHH expression (Innis and Hedera, 2004). To investigate the possibility that Dbf and LSS share a common etiology, we screened five patients diagnosed with LSS for copy number variation at 23 sites between IHH and EPHA4 using multiplex ligation-dependent probe amplification (see Supplementary Information). No copy number variation was detected (data not shown).
3
Discussion
3.1
Ectopic Ihh expression in Dbf/+ is modified in the absence of Shh and is associated with loss of ZPA identity
Although we have previously shown that expression of ectopic Ihh in Dbf/+ limb buds coincides with the onset of limb bud expansion at E10.5 (Crick et al., 2003), it was not known whether the prepattern of Dbf limbs might be affected by the mutation prior to Ihh expression. However, no differences were detected in the expression of Gli3 or Hand2 in wt and Dbf/+ embryos at E9.5 or E10.5, consistent with the hypothesis that ectopic Ihh expression represents the primary pathogenic event. By E11.5, expression domains of both Hand2 and Gli3 were more extensive in Dbf/+ than wt limb buds, suggesting that Ihh signalling is able to modify their expression.
In Dbf/+ limb buds, Ihh and Shh are expressed in discrete adjacent domains. Exclusion of Ihh from the Shh domain is reminiscent of the exclusion of the Hh-inducible gene Gremlin from this domain; Scherz et al. (2004) suggested that the effect may be due to high levels of intracellular autocrine Shh signalling. The loss of identity of the ZPA resulted in a abnormal expansion of the posterior limb bud mesenchyme in Shh−/−;Dbf/+ mice leading to the additional postaxial polydactyly seen in these mutants.
3.2
Abnormal gene expression leading to an aberrant Gli3 ratio underlies Dbf polydactyly
To elucidate the limb patterning underlying Dbf/+ polydactyly and to investigate the generation of the broader, more regular fan of digits seen in Shh−/−;Dbf/+ mutants, we studied the expression of a range of limb patterning and development genes at E10.5. Dbf/+ mutant limbs show an anterior expansion of the positive regulators of Hh signalling Ptc1, Gli1 and the downstream targets Hoxd13 at E10.5 and Hand2 by E11.5. Dbf/+ limbs also show a reduction of Gli3 expression, which is thought to be negatively regulated by Hh signalling. Conversely, due to the complete lack of Hh activity in Shh−/−;Dbf/+ mutant limbs prior to E10.5, Gli3 is ubiquitously expressed in these limb buds until this stage, when it is down-regulated in Shh−/−;Dbf/+ but not Shh−/− mutants. Gli3R is thought to repress expression of Hoxd13 and Hand2 and Fgf4 in the anterior of wt limb buds while Gli3A induces the expression of Gli1 in the posterior region (reviewed in Tickle, 2006). Therefore, the postaxial polydactyly seen in Shh−/−;Dbf/+ mutants may be due to the loss of identity of the ZPA with concomitant posterior extension of the Ihh domain. In contrast, the preaxial polydactyly that is present in both Dbf/+ and Shh−/−;Dbf/+ mutants is correlated with ectopic Gli3A-induced Hh signal transduction together with lack of repression of posterior patterning genes by Gli3R in the anterior of the limb bud. We suggest that the discrepancy between the very low level of Gli3 mRNA (Fig. 4) and the Gli3 protein detected in Shh−/−;Dbf/+ limb buds at E10.5 (Fig. 5) indicates the perdurance of protein after the gene has been down-regulated.
3.3
Identification of the Dbf mutation
The interpretation of the mechanism of the Dbf mutation has been hampered previously by the failure of attempts to identify the underlying mutation. Using a Southern blotting and inverse PCR strategy we have demonstrated that a ∼600 kb deletion underlies the Dbf phenotype. The presence of simple sequences at both breakpoints may have predisposed them to breakage; the lack of significant similarity between the breakpoints (except for a 3 nucleotide identity at the breakpoints themselves) suggests that the rearrangement is likely to have involved nonhomologous end joining (NHEJ). Further analysis of sequence at the breakpoints revealed that the distal breakpoint resides within the degenerate poly(A)n tract of a short retrotransposon (SINE) of the rodent B1 family, which, like human Alu repeats originate from 7Sl RNA (Vassetzky et al., 2003).
The deleted region in Dbf is relatively gene-dense and completely deletes 24 known and predicted transcripts as well as interrupting Nhej1 at the centromeric breakpoint. Several of these genes have previously been implicated in abnormal mouse phenotypes or human disease; information on the known expression patterns and functions of these genes is summarized in Supplementary Table 2. Abnormalities associated with genes in the deleted region may contribute to additional aspects of the heterozygous Dbf phenotype such as the broadened skull, hydrocephalus, reduced viability and fertility, thickened tail and supernumerary hair follicles. However, none of the homozygous null phenotypes resulting from specific targeting of the Ptprn, Des, Inha or Slc4a3 genes is lethal in late embryogenesis so the cause of death at E14.5 in Dbf homozygotes remains unclear. This could be attributable to loss of function of any of the genes within the interval for which homozygous mice have not yet been described, and/or to homozygosity for the ectopic Ihh expression defect. Interestingly a recent study reported a human fetus with a balanced de novo translocation t(2;7)(q36;p22) with the chromosome 2 breakpoint interrupting the orthologue of Nhej1 at a position similar to the start of the Dbf deletion (Cantagrel et al., 2007). The consequence of this translocation, as in Dbf, would be to isolate the human IHH gene from possible regulatory sequences present on the opposite side of the NHEJ1 breakpoint. Although the terminated fetus exhibited syndactyly of all four limbs, polydactyly was not present, suggesting that the translocation did not result in ectopic IHH expression.
We have presented evidence that the prepattern of Dbf limb buds is unaffected and that the preaxial polydactyly is attributable to a reduction in Gli3R resulting from ectopic Ihh expression. It is interesting that preaxial polydactyly, the most striking aspect of the Dbf phenotype, is unlikely to result directly from haploinsufficiency of any of the genes in the deleted region. Rather, the deletion appears to affect a cis-acting regulatory element of Ihh, which could be a repressor located within the deletion, or an enhancer beyond the deleted region. Other examples of regulatory mutations acting at a distance have been reviewed by Kleinjan and van Heyningen (2005). Pinpointing the regulatory sequences involved remains a major challenge, one notable success being the identification of the ZPA sequence regulatory sequence (ZRS) which lies ∼1.0 Mb upstream of Shh and regulates its expression in the ZPA; mutations in the ZRS lead to ectopic Shh expression resulting in preaxial polydactyly (Lettice et al., 2002). However, owing to the large size of the Dbf deletion and the large number of genes and highly conserved non-coding elements within it, it will be challenging to delineate the precise mechanism underlying ectopic Ihh expression in the Dbf mouse.
4
Materials and methods
4.1
Generation and identification of mutant mice
Mice heterozygous for the Shh null allele (Chiang et al., 1996) on the C57BL/6J background were mated to Dbf/+ mice on the 3H1 background. The Shh mutant allele was detected as previously described (Chiang et al., 1996). Homozygous Shh−/− embryos were identified by their phenotype.
To genotype Dbf/+ embryos (prior to the identification of the causative deletion), Dbf/+ mice were crossed with wt Mus musculus castaneus and the Dbf F1 progeny were bred with C3H wt mice. Embryos were genotyped using primers which amplify the marker D1Mit46 located ∼2.3 cM from Ihh (P1 5′-AGTCAGTCAGGGCTACATGATG-3′, P2 5′-CACGGGTGCTCTATTTGGAA-3′). This produces amplification products of 276 bp and 320 bp on the C3H and Mus musculus castaneus backgrounds respectively.
4.2
Whole mount in situ hybridization
Doubly heterozygous Shh+/−;Dbf/+ were crossed with Shh+/− mice and embryos of all six possible genotypes were collected for analysis of gene expression domains and morphology: wt, Shh+/−, Shh−/−, Dbf/+, Shh+/−;Dbf/+ and Shh−/−;Dbf/+. To ensure consistency between developmental stages, only forelimb buds were analysed and a minimum of two samples were examined with each probe. Timing of embryos was by the vaginal plug method: 12.00 noon on the day on which the plug was observed was regarded as E0.5. Pregnant females were sacrificed on the appropriate day by cervical dislocation and the embryos were dissected from the uterus in ice cold phosphate buffered saline (PBS) (140 mM NaC1, 3 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4) followed by immersion in cold tissue fixative in accordance with the Animals (Scientific Procedures) Act, 1986. Where necessary, yolk sacs were removed for genotyping and embryos were fixed by immersion overnight at 4 °C in 4% paraformaldehyde in PBS. Embryos were dehydrated by sequential washing in 25%, 50%, 75% ethanol in PBT (PBS + 0.1% Tween 20) and finally by two washes in 100% ethanol. They were stored at −20 °C until required.
Single stranded digioxygenin-UTP labelled antisense riboprobes were generated from linearized plasmids containing cDNAs. Whole mount in situ hybridization was carried out essentially as described by Wilkinson (1992).
4.3
Skeletal preparations
Embryos for skeletal staining were dissected and fixed in 95% ethanol for 1–3 days. They were immersed in alcian blue stain (75% ethanol, 20% acetic acid, 3 mg/ml alcian blue) for 21–28 days at 37 °C. They were cleared in 0.8% KOH, 20% glycerol. Following clearing, they were sequentially dehydrated and stored in 50% ethanol/50% glycerol.
4.4
Western Blotting
The polyclonal antibody specific for the amino terminus of Gli3 was a gift from Dr. Chin Chiang (Litingtung et al., 2002). Three μg of protein lysate derived from the anterior and posterior halves of ∼6 E10.5 forelimb buds were resolved on 4–12% polyacrylamide gels. Gli3 protein was detected using anti-N-terminal Gli3 (1:300) primary antibody and biotinylated anti-rabbit immunoglobulin-γ secondary antibody (1:1000). Protein bands were visualized by incubation with a streptavidin-peroxidase conjugate followed by an enhanced chemiluminescence detection method (Amersham).
4.5
Characterization of the Dbf deletion
To determine whether genomic rearrangements were associated with Dbf, single copy probes labelled with α32P-dCTP were synthesized and used to hybridize Southern blots of DNA isolated from heterozygous 3H1 (C3H × H101 hybrid) Dbf mice and wt mice from both background strains. We used the mouse genome mm9 sequence release (July 2007) for all analyses presented in this paper. A probe corresponding to 75,099,314–75,099,651 bp revealed the absence of a polymorphic 8 kb SpeI fragment in Dbf, found in the C3H parental strain, suggesting the existence of a deletion. A further Southern blot using a probe corresponding to 75,097,987–75,098,255 bp revealed a 1.2 kb BspHI fragment present only in Dbf. This 1.2 kb breakpoint fragment was isolated by inverse PCR. Briefly, genomic DNA from Dbf was digested with BspHI, diluted to 10 ng/μl and T4 DNA ligase was added to promote intramolecular ligation. Religated DNA was used directly in an inverse PCR using the primer pair: 5′-GCATTTGAGATTGAGACAAGCACTCTCCACAC-3′ and 5′-ACAGCGCTAGACAGAAAGCCTGCTTGCT-3′. DNA sequencing revealed 228 bp of unknown sequence that was shown by BLAST analysis to originate from a region of chromosome 1, ∼596 kb telomeric from the breakpoint. PCR amplification with primers designed either side of the breakpoint (5′-TGGTCTGGAGAGACAGCTCGTCCAGAG-3′ and 5′-GAGTTGAAGAGTTGGCATAGTGGTGCACAC-3′) was employed to confirm the site of the deletion.
To confirm the Dbf lesion was a true deletion, primers were designed to amplify three regions within the deletion predicted to contain polymorphisms variable between the C3H and H101 background strains. These regions were located at ∼150 kb intervals within the deleted region. The primer pairs used were site 1, 5′-GCCCTCATGCTTGAGTACCTTGCCTGTGAT-3′ and 5′-GTCCTCCCAGGGGCTGAGCAGAGTG-3′; site 2, 5′-TAGACTGAGCACCCGGCCTAACATGCTC-3′ and 5′-TGTGTCATCCACCCGGTGCCTCTGACT-3′; site 3, 5′-TAGAATTCCCACTGGGTCCACCCACTC-3′ and 5′-CATACATCCGTGTACATGTACTGACTGTCACTG-3′. Amplification products were digested with appropriate restriction endonucleases to discriminate between the alleles. Site 1 contained a novel TACC insertion polymorphism and was digested with HphI, site 2 contained a known C/T polymorphism (rs31657679) and was digested with AvaI and site 3 contained a known polymorphism (rs3049959) and was digested with Hpy8I. In each case only the H101 allele was present indicating that the C3H chromosome carried the deletion. The presence of both background strains was confirmed on the centromeric side of the Dbf deletion by sequence polymorphisms observed during Southern blotting (data not shown). Both strain backgrounds were shown to be present on the telomeric side of the deletion by AseI restriction digest of a fragment containing a novel informative C/T polymorphism which was amplified using the primer pair 5′-CAACAAAGCCCACATCAATTCACTCAGGCCGTG-3′ and 5′-CACCCTGCCTCAACCTCTCACCTGCTAG-3′. | [
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Eur_Radiol-3-1-2077918 | Multi-detector row computed tomography angiography of peripheral arterial disease
| With the introduction of multi-detector row computed tomography (MDCT), scan speed and image quality has improved considerably. Since the longitudinal coverage is no longer a limitation, multi-detector row computed tomography angiography (MDCTA) is increasingly used to depict the peripheral arterial runoff. Hence, it is important to know the advantages and limitations of this new non-invasive alternative for the reference test, digital subtraction angiography. Optimization of the acquisition parameters and the contrast delivery is important to achieve a reliable enhancement of the entire arterial runoff in patients with peripheral arterial disease (PAD) using fast CT scanners. The purpose of this review is to discuss the different scanning and injection protocols using 4-, 16-, and 64-detector row CT scanners, to propose effective methods to evaluate and to present large data sets, to discuss its clinical value and major limitations, and to review the literature on the validity, reliability, and cost-effectiveness of multi-detector row CT in the evaluation of PAD.
Introduction
Before multi-detector row CT (MDCT) technology was available, the evaluation of peripheral arterial disease (PAD) using CT was restricted to imaging only a portion of the peripheral arterial tree [1–8]. With the introduction of four-detector row CT (4D-CT) in 1998, this major limitation was overcome. A complete coverage of the lower extremity inflow and runoff arteries was possible with one acquisition using a single-contrast bolus. With the launch of the 16-detector row CT (16D-CT), the spatial resolution increased to near isotropic voxels and the contrast medium efficiency improved [9–11]. True isotropic high spatial resolution of the entire volume was possible using the 64-detector row CT (64D-CT) scanner. In addition, improved X-ray tube capacity and scan speed allow submillimeter acquisition of a large coverage without limitations. These developments made multi-detector row CT angiography (MDCTA) an accurate alternative for the assessment of the peripheral arteries [12–25]. Using standardized scanning and reviewing protocols, peripheral CT angiography is a robust non-invasive technique for evaluating chronic and acute disease of the peripheral arteries. We present a review concerning our experience with 4-, 16-, and 64-detector row CT scanners in patients with PAD.
Technique
Preparation
There are no specific prescanning preparations necessary for MDCTA of the peripheral arteries. The patient is placed comfortably to avoid movement, in the supine position with raised arms on the CT table. The legs are stabilized with cushions around the legs and slightly strapped with adhesive tape distally. It is important that the patient does not wear metal zippers or buttons on their clothing, since this can have a negative influence on the image quality, especially when using postprocessed images. Oral contrast should not be used, as this complicates postprocessing display (Table 1). Contrast material needs to be administered at body temperature to decrease the viscosity. The protocol can be completely programmed into the scanner.
Table 1Prescanning preparationParameterDescriptionClothingNo metal parts on clothingOral contrastNoneI.V. cannula antecubitalMinimally 22 G (0.6 mm inner diameter, blue valve)PositioningSupine, stabilized and lightly strapped, feet-first and arms elevatedRespiratory phaseInspiration during abdominal-pelvic range
Technical parameters
The main challenge for peripheral CT angiography is the great range of the vascular system that needs to be depicted. Using a scanogram of approximately 1,500-mm length, the coverage of the acquisition is planned from the celiac trunk (T12 vertebral body) to the level of the talus using 4D-CT, or to the level of the feet using 16D-CT or higher (Fig. 1).
Fig. 1(a) Scout image with three planned reconstruction batches of the abdomen, the upper legs, and the lower legs to preserve postprocessed image resolution. The frames 3-1, 3-2, and 3-3 depict the field of view of the three data sets, which need to be as narrow as possible to optimize pixel size. Whole-body volume maximum intensity projection (MIP) images after semiautomated bone removal of the abdominal data set (b), the femoral data set (c), and the crural data set (d)
Scan duration
The optimal scan duration for peripheral CT angiography varies between approximately 20 to 40 s, depending on the number of detector rows and the collimation (Table 2). The velocity of a contrast bolus to travel from the aorta to the popliteal arteries varies from 29 to 177 mm/s in patients with PAD [26]. This large variability is unpredictable and does not correspond to the severity of PAD. Based on these bolus travel times, it is recommended to limit the maximum table speed on faster scanners to 30 mm/s to avoid outrunning the bolus, leading to poor distal vessel opacification. This can be obtained, for example, by limiting the gantry rotation speed from 0.33 to 0.5 rotations per second or reducing the pitch (Table 2). Moreover, it is advised to program a second acquisition protocol into the scanner to start immediately if delayed distal enhancement is detected (Fig. 2). Because the time of the contrast bolus to travel from the aorta to the ankles varies from 7 to 40 s, a longer scan duration increases the risk of venous contamination, especially when there is critical ischemia and inflammation [26, 27]. Nevertheless, the discrimination of the arteries from the veins is often possible due to the stronger arterial enhancement and the anatomic 3D information [12].
Table 2Acquisition parameters for various multi-detector row computed tomography (MDCT) configurations for the angiography of peripheral arteriesType of scannerSection collimation width (mm)bRotation time (s)PitchcTable feed (mm/ rotation)Table speed (mm/s)Scan duration (s) dCharacteristics4D-CTa4 × 2.50.51.5153040Slow scan protocol, thick minimal slice width16D-CTa16 × 0.750.51.3153040Slow scan protocol, high resolution16 × 1.50.50.7173435Slow scan protocol, less resolution, better in obese patients16 × 1.50.51.0244825Fast scan protocol, less resolution, reduction of contrast media64D-CTa2 × 32 × 0.60.50.8153040Slow scan protocol, high resolution, isotropic voxel, double z-sampling, scanning of obese patients possible2 × 32 × 0.60.330.8164825Fast scan protocol, reduction of contrast media, high resolution, isotropic voxel, double z-sampling, scanning of obese patients possible2 × 32 × 0.60.331.019.86020Fast scan protocol, reduction of contrast media, high resolution, isotropic voxel, double z-sampling, scanning of obese patients possible, risk of outrunning the bolusaProtocols designed for Siemens CT scanners (Siemens Medical Systems, Erlangen, Germany) and should be modified appropriately for other models and manufacturersbValues are number of sections times section widthcPitch as the ratio of the table feed per rotation over the total width of the collimated beamdScan times representing a scanned range of 120 cmFig. 2Images from the first and second delayed acquisition of a 37-year old male with blue toe syndrome of the left hallux. (a and b) VRT images of the first acquisition show in the aneurysmatic abdominal aorta a short occlusion of the left femoral artery (white arrows) due to thrombo-embolism and an occlusion of the entire right superficial femoral artery. The anterior tibial arteries seem occluded in both legs. (c and d) VRT image of the feet with the first and the delayed second acquisition. The first acquisition (c) shows that the arteries of the feet are not enhanced yet due to slow flow (asterisk). The delayed acquisition (d) shows that both dorsal pedal arteries are patent and that the proximal arcuate artery and the first dorsal metatarsal artery (black arrows) of the left foot are occluded due to thrombo-embolism
Using recent MDCT scanners, fast scans (25 s or less) can be performed of the peripheral arteries to reduce the amount of contrast media. To allow fast scan speed using 16-detector row CT (16D-CT) scanners, a wider collimation must be used (Table 2). The 64-detector row CT (64D-CT) even allows to perform fast scans while maintaining submillimeter collimation. However, to ensure distal opacification, the scanning delay must be increased appropriately in fast scans. Another difficulty of a fast scan is that there is a greater risk of asymmetric enhancement in patients with severe unilateral vascular disease. Therefore, it is safer to choose a slower scan speed.
Collimation
To aim for maximal spatial resolution, a thin section collimation width allows a narrow effective slice width. Furthermore, the partial volume effect and blooming effect of calcium will be reduced (Fig. 3) [10]. The collimation should be chosen as narrow as possible but still allowing for a table speed of 30 mm/s and depends on the number of detector-rows and heat capacity. On a 4-detector row CT (4D-CT), the collimation is limited to 4 × 2.5 mm, whereas the 16D-CT and 64-detector row CT (64D-CT) allow a submillimeter collimation of 16 × 0.75 mm and 32 × 2 × 0.6 mm, respectively.
Fig. 3a, bImages of 16-detector row CT (16D-CT) acquired with a collimation of 0.75 mm showing the effect of slice width (SW) on the blooming of the arterial wall calcifications. (a) Reconstructed axial image of the right external and internal iliac artery with SW of 3.0 mm using a B46 reconstruction kernel shows more blooming of the calcifications than (b). (b) Reconstructed axial image of the right external and internal iliac artery with SW of 0.75 mm using a B46 reconstruction kernel with less blooming of calcifications
Using 16D-CT in obese patients, the thin collimation protocol leads to unacceptable noise levels in the abdomen and pelvis because the tube is unable to deliver the necessary dose in this submillimeter configuration. In order to enable the tube to deliver a higher dose, a wider collimation (16 × 1.5 mm) with a reduced pitch factor of 0.7 (Table 2) is used to improve the image quality in obese patients. For the 64D-CT scanner, there is no longer a tradeoff between resolution and scan speed, and it allows, even in obese patients, a fast submillimeter scan protocol.
Contrast injection
It is important in peripheral CT angiography to obtain a high and homogenous enhancement of the arterial tree and to synchronize the acquisition with the enhancement. The optimization of acquisition timing and contrast medium delivery is essential for vascular assessment and image postprocessing. Normally, attenuation values higher than 200 HU in the arteries is considered suitable in MDCTA [12, 13]. For the intravenous injection of contrast medium in the antecubital vein, 22- and 20-gauge intravenous cannulas are needed for the maximal flow rates of 3.5 and 5.0 mL/s, respectively.
Acquisition timing
Due to the interindividual hemodynamic variability in peripheral CT angiography, reliable timing techniques are preferred over using a fixed delay. The test-bolus technique relies on the dynamic monitoring of small contrast boluses to measure the contrast arrival and travel time at the proximal and distal arteries, respectively. The bolus-triggering technique is a commonly used timing technique that is based on repetitive low-dose sequential scans at the level of the abdominal aorta, to monitor the arrival time of the contrast media. The acquisition starts automatically when the preferred threshold is reached, approximately 100 to 150 HU above the baseline value. During a transition delay, which is the time needed for the table to move and start the scan, of approximately 4 s, breathing instructions can be given to the patient. During this delay, the enhancement of the aorta will further increase to an absolute value of more than 200 HU.
For a fast scan protocol, an extra delay must be added to the contrast arrival time to ensure distal arterial opacification [28]. This extra delay can be calculated as 35 s minus the scanning time. Thus, for a scan time of 25 s, a extra delay of 10 s. must be added. Another option is to monitor at the proximal level of the popliteal artery and to start the scan manually when enhancement is visualized. Consequently, the time of contrast arrival increases by approximately 8 s [26, 27] and the transition delay of the scanner increases to 11 s to travel from the knees to the diaphragm and then starting the acquisition.
Contrast injection
The volume of contrast material ranges from 120 to 160 ml for a typical scan duration of 40 s. The amount of contrast media depends on the scan duration and on the flow rate. Because the last volume of the bolus will not contribute to the enhancement when scanning below the knees, the injection duration can be shortened by 5 s, e.g., a 35-s injection time is used for an acquisition of 40 s. However, to ensure the enhancement of all arteries, the injection duration should not be shorter than 30 s and in fast scan protocols, a delay time needs to be added appropriately to prevent outrunning the contrast bolus. A flow rate of 3 to 4 ml/s is necessary for adequate arterial enhancement [12]. This corresponds to an iodine administration rate of 1.0 to 1.4 g/s using a contrast media concentration of approximately 320 to 350 mg I/mL. Based on the reported literature the average values of contrast media volume, concentration, injection rate, and administration rate are 134 ml, 341 mg I/mL, 3.5 ml/s, and 1.2 g/s, respectively [9–25, 29–36]. By increasing the iodine concentration to a concentration of 400 mg I /mL, the iodine administration rate can be increased to 1.6 g/s to increase the enhancement [37]. To optimize the enhancement, 20 to 60 mL of saline is injected immediately after the contrast media. A tighter bolus can be obtained to increase the attenuation.
Using a monophasic injection rate, the arterial enhancement increases over time to decrease at the end of the bolus. Consequently, the Hounsfield values of the enhanced arteries start lower at the level of the aorta and increase at the level of the popliteal artery to the highest attenuation value, and, subsequently, decrease distally in the runoff arteries, especially for longer scan durations [26]. A more homogenous enhancement can be achieved using a biphasic injection rate using a higher rate (5–6 ml/s) at the beginning (during the first 5 s) of the injection and a lower rate (3 ml/s) for the remaining volume. In clinical practice, a monophasic injection rate is often used because it is a simple method and has resulted in adequate image quality [37].
Patient dose in MDCT
A particular concern with MDCT scanners is delivering potentially higher radiation doses. To maintain the noise level in submillimeter slices, the dose needs to increase proportionally. On the other hand, with the increasing number of detector rows, the z-axis efficiency improves, since the overbeamed area decreases. Current MDCT scanners present an indication of patient dose on the scanner console for dose awareness and to help optimize the scan protocol. Useful in CT angiography is that, when reducing the X-ray energy, the contrast-to-noise ratio increases. Compared to a standard scan with 120 kVp, selecting 100 kVp, results in a dose saving of approximately 40% [38–40]. Furthermore, dose reduction can be achieved by decreasing the tube current using automatic tube current modulation. With angular tube current modulation, the tube current varies during the course of a rotation. The changing attenuation through different projections around the patient (e.g., at the level of the pelvis) can be used, to reduce unnecessary x-rays in the anterior–posterior projection without any substantial effect on image quality [9, 41, 42]. With longitudinal tube current modulation, the tube current varies along the z-axis based on the size, shape, and attenuation to maintain a predefined noise ratio. Compared with constant tube current, this technique results in acceptable image noise and a dose reduction of 20% or more without compromising diagnostic image quality [9, 41, 42].
The average patient dose reported in the literature in the assessment of PAD with CT angiography is 7.47 mSv [9, 12, 24, 31, 43]. The radiation risk from these doses is not a major concern in patients with PAD. Their life expectancy is shorter than the latency period of a radiation-induced fatal malignancy [44–46].
Display and evaluation
Image reconstruction
The raw data set is reconstructed using an increment with 50% to 70% overlap. Peripheral CT angiography generates more than 1,500 axial images, depending on slice width and reconstruction increment. It is recommended to reconstruct separate data sets. Routinely, we calculate three separate data sets of the peripheral runoff (Fig. 1). The first advantage is that it allows us to reconstruct thicker slices, e.g., of 1.5 mm for the abdominal and femoral data set, and thinner slices for the crural data set to optimize the resolution and to minimize the data load [9, 10, 16, 20, 23]. Secondly, longitudinal images that are calculated from the entire data set have a decreased resolution, due to the limited display matrix (e.g., 512 × 512) [32]. Images that are reconstructed from the separate data sets will preserve the initial longitudinal resolution.
A smooth kernel (B20 for Siemens CT scanners) is generally used in CT angiography and leads to an accurate depiction of the diameter of the vessels and is very appropriate for postprocessing. A sharp kernel (B46) is used when stents or severe vessel wall calcifications are present, as it minimizes the blooming effect at the cost of some increase in the noise level [47].
The field of view (FOV) is selected as small as possible to optimize pixel size. A FOV of 380 mm, 350 mm, and 300 mm for the abdominal, femoral, and crural data sets, results in pixel sizes of approximately 0.74 mm, 0.68 mm, and 0.58 mm, respectively. Also, the FOV can be further decreased to 200 mm by including only one leg, leading to a pixel size of 0.4 mm.
Advanced postprocessing and image evaluation
Additional two-dimensional (2D) and three-dimensional (3D) postprocessing techniques are required to facilitate interpretation and presentation. Reviewing exclusively the transverse images is inefficient and less accurate than reviewing a combination of reformatted images.
To preserve the study quality for clinical decision making, a standard set of postprocessed images needs to be included in the protocol. These include thin-slab maximum-intensity projections (MIPs) through visceral and renal arteries and the abdominal aorta, through femoropopliteal arteries, and through crural arteries (Fig. 4); whole-volume MIPs of the separate data sets after bone removal (Fig. 1) and when necessary, after removal of vessel wall calcifications (Fig. 5); and curved planar reformations (CPRs), e.g., through the iliac arteries. Volume-rendered (VR) images are fast and effortless created to present the pathology to clinicians, who normally do not have the possibility to review the data set interactively.
Fig. 4a–dStandard slab MIP images in the postprocessing protocol make MDCTA of peripheral arteries on a routine basis feasible. Slab MIPs are easy and fast to create and to evaluate. The images depict the vasculature without superimposing bones (a and b). From the abdominal data set, MIP images are created in coronal projection to depict the renal arteries (b) and in sagittal projection to depict the celiac trunk and mesenteric arteries (d). The aorta is also depicted for evaluation. (c) Standard coronal slab MIPs from the data set of the upper legs are created, which are parallel to the superficial femoral and popliteal artery. (d) Standard coronal slab MIPS from the data set of the lower legs display the crural arteriesFig. 5a–dVolume MIP images in anteroposterior projection show the result of three different threshold levels used for the segmentation of arterial wall calcifications. (a) Volume MIP before removal of the calcifications shows that the lumen is not visible. (b) Volume MIP after removal of the calcifications shows that, still, many voxels of calcification are present, hampering lumen assessment (arrows). (c) Volume MIP shows angiogram after calcium segmentation using a correct threshold level allowing lumen assessment. The rest of the voxels of the burden of calcifications are just visible as unesthetical noise, which is, however, preferable to introducing pseudo-stenoses (d) (arrows) by using a too low threshold level
The data sets are reviewed effectively by evaluating the standard set of postprocessed images and, interactively, exploring the data set using multiplanar reformations (MPRs) . The transverse images (or true cross-sectional images) need to be considered to verify diseased segments [4, 6, 19].
When extensive calcifications or stents are present, the vessel lumen visibility and the clinicians’ confidence in the CT images will decrease [25]. In whole-volume MIPs, superimposing calcifications can be selected to be removed digitally from the data set using thresholding and region-growing techniques (Fig. 6). However, the removal of the numerous arterial wall calcifications can be very time consuming. Another limitation of these segmentation techniques is that readers should be aware of artificial stenoses and occlusions. These can be introduced when voxels that represent lumen are inadvertently removed when in close contact with the bones (Figs. 7 and 8) or when a too low threshold value is used (Fig. 5). In addition, in VR images, the lumen is also obscured by vessel wall calcifications and, as a result, should not be used for the lumen assessment (Fig. 6). A more reliable technique for stenosis detection in extensive calcified arteries is CPR, which displays the lumen as a longitudinal cross-section (Fig. 6). When using an application that semi-automatically traces the vessel lumen, the risk of an inaccurately positioned central lumen line is minimized. The CPR projection should include at least two perpendicular longitudinal projections and true cross-sectional images can be viewed for lumen assessment [19]. Software tools are available for automatic quantitative evaluation of the traced lumen and to generate a graphical presentation of luminal diameter (Fig. 9). Multipath CPRs are under development and could enhance image evaluation.
Fig. 6a–dInfluence of vessel wall calcifications on postprocessed images and the ability for lumen assessment. (a) VRT image (medial view) of right femoropopliteal segment showing arterial wall calcification; does not allow luminal assessment. (b) CPR image (anteroposterior view) shows the interior of blood vessels as a longitudinal cross-section, even in the presence of the arterial wall calcifications. This is the preferred imaging technique when extensive calcifications of the vessel wall are present. Volume MIP (anteroposterior view) after bone removal using region-growing and threshold techniques (c) does not allow lumen evaluation. Volume MIP after additional calcification removal (d) removes superimposing calcification to enable lumen evaluationFig. 7a–cImages of segmentation artifacts due to bone removal in 16D-CTA. (a) Volume MIP after bone segmentation of the lower legs showing a pseudo-occlusion of both distal anterior crural arteries (arrows), which is caused by segmentation of the bones. (b) MIP of the lower legs showing the anterior tibial arteries in close proximity to the tibia (arrows), which is the cause of the false positive pseudo-occlusion. (c) Axial image of the lower legs just caudal from the pseudo-occlusion, showing the patency of both anterior tibial arteries of both legs in close proximity of the tibia (arrows)Fig. 8a–cApplying blue color to the voxels selected for removal helps to identify the sites of segmentation artifacts in VRT images. (a) VRT image before bone segmentation of the lower legs showing patent proximal anterior tibial arteries. (b) VRT with blue bones to indicate the voxels to be removed shows the voxels of the bone which are in contact with the proximal anterior tibial artery are not selected for removal and shows the voxels of the artery which are selected for removal. (c) Segmented VRT image showing the pseudo-occlusion of the anterior tibial arteryFig. 9a, bResults of semiautomated quantitative lumen assessment in aortoiliac arteries of a patient with in stent thrombosis. (a) Graph (upper section) displaying the maximum and minimum diameters of the lumen to quantify stenosis. CPR (lower section) through the aortoiliac arteries, which can be rotated around its longitudinal axis, depicts the luminal obstruction (asterisk) due to a thrombus inside an iliac stent. (b) Corresponding transverse image confirms the occlusion of the iliac stent
Wall calcification problem
The depiction of vessel wall calcifications using MDCT can be valuable, since severely calcified arteries may have consequences for bypass surgery. On the other hand, these wall calcifications are known to hamper the assessment of the lumen [2, 10, 14, 19]. Approximately 20% to 50% of the vascular segments contain wall calcifications, of which, 10% severely calcified [11, 19]. Patients with a history of diabetes mellitus, cardiac disease, or elderly age are very likely to have extensive calcifications [48]. Furthermore, we found that patients with Fontaine stage III/IV have more infrapopliteal arterial wall calcifications compared to stage IIb.
How can we deal with the vessel wall calcifications depicted with MDCTA? It is important to use a wider window width (WW) and higher window center (WC) level settings from the usual CT angiography level of around 150 WC ± 250 WW to 200 WC ± 1000 WW for a better differentiation of calcifications and stents from the enhanced lumen and to minimize the effect of blooming. A further minimization of blooming is reached by using a sharper reconstruction kernel and higher spatial resolution.
Especially in MIP images, the lumen is hidden by the circumferential calcifications. In these circumstances, transverse images, CPR images, and the digital removal of the calcifications help to depict the lumen, at least for the larger arteries. Despite all of the available tools, in particular in the smaller crural arteries, the concentric calcifications still hamper lumen assessment [11]. Recent publications showed that a subtraction technique using two acquisitions is feasible in some patients with PAD using MDCTA [24, 49]. In the near future, automated 3D applications could help to minimize the impediment of the calcifications [50]. Whether dual-energy CT angiography can improve this limitation of CT needs to be evaluated.
Clinical value
Because MDCT angiography for the imaging of the peripheral arteries is a rather new non-invasive technique, there are a small number of studies published on its performance and reproducibility (Table 3). The majority report on 4D-CT; two authors report on 16D-CT. There are no reports of the assessment of PAD using 64D-CTA. In our meta-analysis soon appearing in Radiology, which included 436 patients and 9,541 arterial segments, a pooled sensitivity and specificity for detecting a >50% stenosis of 92% and 93% was estimated, respectively.
Table 3Validity of CT angiography in peripheral arterial disease (PAD)AuthoraNo. of patientsNo. of analyzed segmentsNo. of detectorsReported sensitivity (%)eReported specificity (%)eAssessed segmentsStenosis category (%)hRichter et al. 199432ns184nsIliac>50Lawrence et al. 1995613419396Femorocrural>50Raptopoulos et al. 19963962419396Aortoiliac85–99Rieker et al. 199650400173–88b94–100bFemorocrural75–99Rieker et al. 19973021019399Aortoiliac75–99Kramer et al. 199810ns294nsIliocrural>90–99Ishikawa et al. 199949ns19795Bypass graftsnsBourlet et al. 20002231819590Aortoiliac>50Puls et al. 20013118648986Total tree50–99Willman et al. 20034676949199Aortoiliac graftsnsOfer et al. 20031841049192Total tree>50Heuschmid et al. 200318568491c92cTotal tree>50Martin et al. 2003411,31249297Total tree75–99Catalano et al. 2004501,14849693Total tree>50Mesurolle et al. 20041616829193Total tree>50Ota et al. 20042447049999Total tree>50Poletti et al. 2004d12144482/96gnsns>50Portugaller et al. 20045074049283Total treearea >70Romano et al. 2004423,40249395Total treensRomano et al. 2004221,78249294Total treensStueckle et al. 200452ns482100Total treensEdwards et al. 2005441,02447993Total tree50–99Fraioli et al. 2006751,425496–99h94–96hTotal tree50–99Schertler et al. 200517170169690Popliteocrural>50Willmann et al. 2005391,365169696Total tree>50Unpooled mean9194aBased on references [1–6, 8–10, 13–24, 31, 34, 35, 56, 57]bFor various anatomic levelscCalculated from the datadBased only on subtracted MDCTA images, the positive predictive value was 95%eSensitivity as published or calculated overall mean.fDiameter stenosis is mentioned unless specified (>50 means stenosis more than 50% including occlusion)gFor subtracted and nonsubtracted segments, respectivelyhDepending on the MDCTA protocol with varying mAs
Publications on the reproducibility of CT angiography reported a good intertest agreement between MDCTA and DSA (Table 4) and a good to excellent interobserver agreement for 4D-CTA [12, 21, 23, 35] and 16D-CTA (Table 5) [9, 11]. A few studies provide stratified data on the aortoiliac, femoropopliteal, and crural tract and show that the accuracy and reproducibility of the crural tract is lower than for the aortoiliac and femoropopliteal tracts [9, 11, 20, 22, 23].
Table 4Intertest agreement between CT angiography and digital subtraction angiography in PADAuthoraNo. of patientsNo. of assessed segmentsNo. of detectorsReported intertest agreementdAssessed segmentsRaptopoulos et al. 199639624190%AortoiliacBeregi et al. 199720521100%PoplitealTins et al. 200135219184%AortoiliacWalter et al. 2001224564κ = 0.68 (0.50–0.97)cTotal treeRubin et al. 2001183514100%Total treeHeuschmid et al. 2003231,136486%Total treeOfer et al. 200318444478%Total treeRomano et al. 2004423,4024κ = 0.68; 90%Total treeRomano et al. 2004221,7824κ = 0.68; 90%Total treeaBased on references [3, 7, 12, 14, 16–18, 35, 55]bBased on 97% of the segmentscAverage of the reported kappa values (ranges) of the individual anatomical segmentsdAn unweighted kappa statistic (κ) is reported for percentage agreementTable 5Interobserver agreement of CT angiography in PADAuthoraNo. of patientsNo. of analyzed segmentsNo. of detectorsReported interobserver agreementbAssessed segmentsRieker et al. 1997302101ρ=0.95AortofemoralWalter et al. 2001224564κ = 0.71–0.76cTotal treeTins et al. 200135219178%AortofemoralMartin et al. 2003411,3124κw = 0.84Total treeRomano et al. 2004423,4024κ = 0.84;0.86dTotal treeRomano et al. 2004421,7824κ = 0.85, 0.88, κ = 0.80eTotal treeCatalano et al. 2004501,1374κ = 0.80Total treeOta et al. 2004244704κ = 0.88IliacPortugaller et al. 2004507404κ = 0.81Total treeKock et al.f732,2684κw = 0.84Total treeOuwendijk et al. 2005792,41916κw = 0.85Total treeWillmann et al. 2005391,36516κ = 0.85–1Total treeaBased on references [4, 7, 9, 11, 15, 17–19, 21, 23, 35]bAn unweighted kappa statistic (κ) is reported, unless indicated (κw=weighted kappa statistic; ρ=intraclass agreement coefficients as a measure of agreement for ordinal or quantitative data). A linear weighting was used, except in one paper [15], where a quadratic weighting was usedcRange of kappa values of the individual anatomical segmentsdFor reader one and two, respectivelyeFor intraobserver (two readers) and interobserver agreement, respectivelyfBased on unpublished data
MDCTA leads to adequate decision making for treatment recommendations concerning both the anatomical level and the technique of revascularization [51]. A cost-effectiveness study showed that MDCTA is a cost-effective diagnostic strategy in the work-up of PAD [52, 53]. Randomized controlled trials confirmed that MDCTA in PAD is the optimal diagnostic imaging technique [25, 54] and reduces the diagnostic costs when compared to DSA and CEMRA with comparable clinical utility and patient outcomes. Besides these evidence-based results, local expertise and availability also define which modality to use in clinical practice (Table 6).
Table 6Advantages and limitations of multi-detector row CT angiography (MDCTA), contrast enhanced MR angiography (CEMRA), and digital subtraction angiography (DSA) MDCTACEMRADSAIntermittent claudication (Fontaine II)+++Chronic critical ischemia (Fontaine III or IV)−++Short examination time+−−Short postprocessing time−++Outpatient setting++−Availability+−+Non-invasive technique/patient comfortb++−Low diagnostic imaging costs+−−Contrast media tolerance−+−Three-dimensional imaging++−Non-interference of stentsc+−+Radiation risk+(−)d−+(−)dAcute clinical setting+−+Hemodynamic assessment−−a+Extraluminal pathology visualization+−a−aIs only possible when using additional sequencesbFrom [58]cFrom [59]dNegligible risk in population with chronic obstructive PAD
It is reported that arterial wall calcifications lead to false-positive interpretations and a decreased reproducibility in reading MDCTA [2, 14, 11]. We have to acknowledge this limitation with current technology. A preferential indication for MDCTA in patients with intermittent claudication (Fontaine stage IIb) is clearly justified. However, patients with critical limb ischemia (Fontaine stage III/IV), who are likely to have extensive calcifications of the smaller arteries, could be better off undergoing contrast-enhanced magnetic resonance angiography (CEMRA) or digital subtraction angiography (DSA).
Finally, MDCTA is an accurate technique to evaluate the patency after revascularization procedures [43]. The technique can be used in the evaluation of acute ischemia, e.g., after a revascularization procedure or in thrombo-embolic disease (Fig. 10). For aneurysmatic popliteal artery disease or entrapment syndromes of the popliteal artery, MDCTA is the preferred imaging modality (Fig. 11) [55].
Fig. 10a, bAcute thrombosis of the crural arteries in a 53-year-old woman with an acutely cold left leg after stopping anticoagulation therapy. The patient refused angiography. (a) VRT image (posteroanterior view) of MDCTA at the level of the crural arteries shows abrupt stoppage of arterial opacification in the left peroneal, anterior, and posterior tibial artery (arrows). The contralateral right crural arteries are patent. (b) Selective anterograde DSA image (posteroanterior view) confirms the occlusions of the three left crural arteries (arrows) due to thrombo-embolismsFig. 11a–dA 56-year old male patient who had a history of deep venous thrombosis with intermittent claudication of the right lower extremity. (a and b) Thin MIP image shows an aneurysmatic right popliteal artery with a tight stenosis distally. (c) VRT and volume MIP (d) confirm these findings and show patent proximal crural arteries
Conclusion
Multi-detector row CT angiography (MDCTA) is an outstanding non-invasive imaging test in the evaluation of patients with peripheral arterial disease (PAD) and is currently the modality of choice in patients with intermittent claudication. The technique can be used in the evaluation of patency after revascularization procedures and in acute ischemia. MDCTA has been shown to have high diagnostic performance and reproducibility in evaluating peripheral arterial disease (PAD). MDCTA reduces diagnostic costs and provides adequate information for decision making. The most important drawback is the limited lumen evaluation of extensive calcified arteries. MDCTA appears to be clinically less valuable in critical limb ischemia because of extensive crural artery calcifications. | [
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Graefes_Arch_Clin_Exp_Ophthalmol-4-1-2206250 | Ophthalmologists, suicide bombings and getting it right in the emergency department
| Background The number and extent of worldwide suicide attacks has risen sharply in recent years. The objectives of this retrospective study are: to determine the prevalence and outcome of the victims who sustained ocular injury, to describe the activities of ophthalmologists in the setting of an emergency department (ED) receiving mass casualties of a suicide bombing attack and to illustrate some of the treatment obstacles that they encountered and the protocol.
The number and extent of worldwide suicide attacks has risen sharply in recent years [1, 2]. The perpetrators typically mingle among crowds of civilians and detonate an explosive device that is usually strapped on their bodies with the intent of sacrificing their own lives in order to cause the death of as many others as possible. The injuries sustained by survivors of these well-planned attacks combine the lethal effects of penetrating trauma, blast injury, and burns [3]. Suicide attacks on civilians were historically confined to a limited number of countries, but the outrageous and devastating destruction of the Twin Towers in New York City on 11 September 2001 and the bombings in London and Madrid established the universality of such terrorism. With the rise in terror-related activities in urban settings, ophthalmologists worldwide may find themselves treating ocular trauma under conditions unlike any they had experienced before, and certainly remote from the relatively orderly setting of an emergency room in which traumatic ocular injuries can usually be counted on one hand at any given time.
Since the beginning of the latest Israeli-Palestinian Intifada on 29 September 2000, more than 4,600 people—mostly civilians—have been killed or injured by suicide bombings in that area [4]. The Tel Aviv Sourasky Medical Center (TASMC) is the largest hospital in the Tel Aviv metropolitan area. As such, most of the victims of the suicide bombing attacks in its catchment area are evacuated to TASMC, and a special treatment protocol has evolved for coping with the unusual logistic needs of such events. The objectives of this retrospective study are: to describe the activities of ophthalmologists in the setting of an ED receiving mass casualties of a suicide bombing attack, to illustrate some of the treatment obstacles that they encountered and the protocol that evolved for overcoming them, and to determine the prevalence and outcome of the victims who sustained ocular injury.
Methods
IRB approval was obtained for this retrospective interventional case series study. Clinical data on all casualties evacuated to the TASMC due to suicide bombing-related injuries were collected from the trauma registry records and reviewed. Their demographic data were obtained from the main admitting office records.
The senior surgeon stationed at the ED entrance is rapidly provided essential information on the type/location of injuries from the arriving ambulance’s paramedical personnel. He/she pages the designated on-site specialist according to prioritization for urgent management.
Ocular injuries are defined as any blunt, penetrating, or perforating trauma or blast-related damage to the eye, orbit, or ocular adnexa. The terms we used to describe ocular injuries conform to the recommendations the United States Eye Injury Registry and the International Society of Ocular Trauma [5]. Individuals with nonpenetrating or non-lacerating ocular injuries, non-penetrating debris in their eyelids, or superficial burns of the eyelids were excluded from this study, as were victims who died of their injuries before undergoing ophthalmologic treatment for whatever ocular damage had been suffered.
The ocular injuries for each eye were categorized according to type: it was possible to have multiple occurrences of the same type (i.e., multiple corneal lacerations) and of several types (i.e., corneal laceration and retinal detachment) in the same eye.
All records of ocular- and orbital-related trauma that were documented in the trauma registry were collected and analyzed together with hospital and outpatient clinic records. The analysis included age, gender, mechanism of injury, anatomic site of injury, Injury Severity Score (ISS) [6], length of stay, length of intensive care unit stay, and surgical procedures. Ophthalmic information included the initially diagnosed ocular condition, all surgeries performed during the ED stay and afterwards, and final ocular test findings. The data were entered using Excel spreadsheet (Microsoft Office) and a simple descriptive statistical analysis was performed.
Results
There were 13 suicide bombing attacks in the Tel Aviv metropolitan area between October 2000 and October 2004. A total of 352 patients were evacuated to the TASMC ED, and 198 of them were hospitalized. The other 154 patients suffered from minor injury or shock for which they were given appropriate treatment and instruction and sent home. The overall severity of suicide bomb-related trauma was very high: the mortality rate was 8.4% when the attack occurred in open spaces, 15.5% in closed spaces and 20.3% when the bomb exploded inside a bus. The ISS was 1–14 for 74% of the patients (non-hospitalized) and ≥16 for the remaining 26% of the patients (admitted to hospital). One of the prominent hallmarks of suicide bombing injuries is the extremely high prevalence of head injuries: among our patients, 49% suffered from head and neck injuries, 9% head and extremity injuries, 4% head and torso injuries, and 38% torso and other injuries (all data are taken from the experience with the 13 bombings in Tel Aviv).
Seventeen patients (4.8%) were listed in the trauma database as having any ocular or periocular trauma, and several had more than one type of injury. The types of recorded trauma were: open globe injuries (n=7), closed globe injury consisting of severe subconjunctival hemorrhage (n=2), partial thickness lamellar laceration of cornea (n=8) (lamellar flap) of which five were burn-related and three were due to small foreign bodies, and extraocular injuries (n=6), which included three orbital fractures due to primary blast injury and three eyelid lacerations. Primary repair of open globe was performed in six eyes that underwent primary closure of laceration. One patient who was diagnosed during the initial triage as suffering from open globe injury died during the initial trauma surgery, thus no ocular procedure was performed. Two other eyes underwent primary exploration of subconjunctival hemorrhage that was suspected as being open globe due to massive subconjunctival: no laceration was found intraoperatively, and contusion was diagnosed. The one eye that was found to be unsalvageable underwent primary enucleation.
After the initial eye surgery, two patients died from their other injuries within 24 h of the explosions.
Of the eight patients with partial thickness lamellar laceration of cornea, three were discharged and given instructions to return as outpatients on the following day. Five other patients who required and received medical treatment for non-ocular-related medical problems were hospitalized and continued eye treatment as inpatients. All patients in which superficial burns were found (n=5) were treated by manual removal of corneal foreign body and antimicrobial drops, and their recovery was uneventful.
Secondary surgical intervention was performed on four patients (all surviving patients who initially underwent primary closure of open globe) within 1 week of the initial trauma: large intravitreal foreign bodies were extracted in three of them, and an intra-lenticular foreign body was extracted from the fourth (Table 1). At final follow-up (≥2 years post-trauma), the visual acuity of the three patients who suffered from large intravitreal foreign bodies were finger counting (FM)-hand movement (HM). The silicone oil was removed in two of these patients and retained in the third; two of them are wearing large cosmetic contact lenses due to corneal opacities that were cosmetically disturbing. The patient who underwent surgery due to intra-lenticular foreign body and penetrating keratoplasty had final visual acuity of 6/12 (Table 1).
Table 1Relevant data of suicide bombing survivors who sustained open globe injuryNo.Age, years/sexPrimary surgical interventionSecondary surgical interventionForeign body extractedFurther surgeryFinal outcomeFinal visual acuity117/fClosure of open globePatient died within 24 h of trauma216/fNonePatient died within 24 h of trauma362/mEnucleation417/fClosure of open globeLensectomy, vitrectomy, removal of foreign body, endolaser silicone oil injectionMetal shrapnell (5 mm)Silicone oil removalPreserved globe flat retinaFC518/fClosure of open globeLensectomy, vitrectomy, removal of foreign body, endolaser silicone oil injectionMetal ball (2 mm)Preserved globe flat retinaHM619/fClosure of open globeLensectomy, vitrectomy, removal of foreign body, endolaser silicone oil injectionGlass fragment (3 mm)Silicone oil removalPreserved globe flat retinaHM727/mClosure of corneal perforationPenetrating keratoplasty, lensectomy, vitrectomy endolaser, intraocular lens implantationGlass fragment (1 mm)6/12FC, finger counting; HM, hand movement
Discussion
The dynamics of an emergency receiving center of victims of a suicide bombing attack are alien to most ophthalmologists. Today, the setting is characterized by large numbers of victims who sustain injuries that are more complex and more severe than those that had occurred during earlier periods of terror activity [7, 8]. Importantly, suicide bombings are more likely to occur in closed spaces, unlike other mass trauma scenarios such as car bombs, train wrecks, and other outdoor explosions: over 62% of injuries that occur in closed spaces are to the face, head, and neck, thus posing a far greater risk to the ocular structures.
In our experience, 17/352 (4.8%) of the survivors of suicide bombing attacks sustained eye injuries, and 9/17 (52%) required urgent attention. This rate is surprisingly lower when compared to previous reports, which documented that close to 10% of survivors of terrorist blasts have significant eye injuries [13]. We have no explanation for the low rate, but we can speculate that because of their close proximity to the hospital together with the efficiency of the Israeli Magen David (Red Cross), almost all survivors are speedily brought to the hospital for examination after terror-related episodes and are listed as admissions in the trauma records, even those with no complaints or only minor ones. Thus, the large number of admitted individuals artificially decreases the percentage of eye victims. Importantly, 41% of all of the reported ocular injuries [9–13] were severe in degree.
The final visual outcome of all the surgeries we performed was poor: globe preservation was successfully achieved in most cases (6/7), but only one patient with an intra-lenticular foreign body had useful vision postoperatively.
In order to provide the best treatment in such a complicated setting, special adaptations must be made to the treatment algorithm of the ophthalmology team. In terms of individual trauma cases, the victim of a suicide bombing attack is no different from any other eye trauma patient. The sudden presentation of large numbers of injured patients, however, presents two types of challenges: the logistical one of rapidly processing masses of casualties through the system and the medical one of providing the best possible trauma care to severely wounded patients [14]. According to our protocol, all patients who complain of eye symptoms and all unconscious patients who sustain head or face injuries must be checked by an ophthalmologist. This requires special disaster on-call lists of ophthalmologists who are able to arrive to the hospital on extremely short notice since, thanks to the highly efficient organization of our Red Shield ambulance facilities, blast victims usually arrive at the hospital within minutes and are hurried to either diagnostic tests or directly to operating theaters. Upon arrival to the ED, every victim of a terrorist attack is triaged by a senior surgeon who synchronizes the activities of the multifaceted operation. The ophthalmologists already present in the hospital and the ones on-call who arrive to the ED are in contact with that surgeon in order to expeditiously locate the victims with ocular injuries, examine them, and send them to the operation theaters, intensive care units, imaging studies or home. There is a directive in our department that all available staff members must contact the hospital immediately upon learning about any suicide bombing attack to check whether their services are required. In the event of large-scale attacks, they are prepared to be recruited to assist in triaging and in treating all the victims, not just those with ocular injuries. The triage procedure is the key to the successful management of large trauma events: the most important rule is that all patients must be checked by the ophthalmologist wherever they are located on the hospital premises. Trauma patients invariably require urgent treatment and some are sent directly from the ER to either imaging units or immediately to the trauma surgical unit. The senior ophthalmologist on the premises must contact trauma registration services, get a list of all admitted patients (usually assigned numbers upon admission) and make sure that each and every one of them is examined, even during emergency surgery or during imaging interventions for non-ophthalmological injuries. When an open globe is suspected, the eye is immediately patched, and the finding is reported to the surgeon in charge of the patient: the staff is instructed not to intervene in the treatment of the eye. Further evaluation is done only when it is certain that there is no danger of expulsive hemorrhage.
Only patients suspected as suffering from open globe injury undergo urgent primary closure of the wound. Since that patient invariably presents with multiple injuries and may not be fit for transfer to the ophthalmology operating theater, however, special alterations to the surgical protocol may be required. For instance, no ophthalmological microscope is available in our trauma center because space is limited in the trauma room due to the concomitant performance of many surgeries and given the cumbersome structure of an ophthalmic microscope. There is, however, a high-quality neurosurgical microscope that has a long arm that can be placed at sufficient distance from the patient and the life-support machines so that it can be used without disturbing the anesthesiologists and other trauma teams as they work, and this microscope is used with great success during primary closure. Other eye surgery procedures are postponed, either until the patient has been stabilized or they are scheduled for a later date. This highlights the first critical responsibility of the ophthalmologist in the mass trauma setting, that of identifying which surgical procedures must be carried out immediately. The order of surgical intervention deserves special attention: due to the characteristic complexity of the injuries, most of the patients required multiple procedures immediately following the trauma. The established protocol adopted among our surgeons is (in descending order): trauma surgery (for life-threatening conditions, performed by either trauma surgeons or neurosurgeons), ophthalmologic interventions (immediate surgery or instructions for palliative care), and orthopedic and plastic surgery interventions.
Finally, terrorist bombings present a danger to the ED staff members that is never associated with any other mass casualty situation: there is a very real chance of explosion by a second-hit, either by explosive material remaining on the perpetrator’s body, or, even more threatening, a second suicide bomber who infiltrates the ED disguised as one of the victims and detonates the bomb inside the crowded ED. Thus, a unique caveat in the ED protocol for terrorist bombing attacks is heightened vigilance, starting from the chaotic first minutes after the arrival of the victims.
Providing medical assistance in an ED to victims of suicide bombing attacks is a harrowing experience: physicians who work in an urban hospital are more and more likely to be exposed to such events [14]. In his excellent editorial, Hirshberg wrote "Urban terrorism, the scourge of the 21st century, is already at our doorstep and surgeons are called upon to play leadership roles in shaping the emergency response in their hospitals. Learning from the experience of those for whom the unthinkable has become a daily reality can help us develop and implement more effective answers to the threats in our own communities" [14]. | [
"terror",
"ocular trauma",
"vitrectomy",
"intra ocular foreign body"
] | [
"P",
"P",
"P",
"M"
] |
Int_J_Biochem_Cell_Biol-2-1-2267855 | Site-directed mutagenesis of Arginine282 suggests how protons and peptides are co-transported by rabbit PepT1
| The mammalian proton-coupled peptide transporter PepT1 is the major route of uptake for dietary nitrogen, as well as the oral absorption of a number of drugs, including β-lactam antibiotics and angiotensin-converting enzyme inhibitors. Here we have used site-directed mutagenesis to investigate further the role of conserved charged residues in transmembrane domains. Mutation of rabbit PepT1 arginine282 (R282, transmembrane domain 7) to a positive (R282K) or physiologically titratable residue (R282H), resulted in a transporter with wild-type characteristics when expressed in Xenopus laevis oocytes. Neutral (R282A, R282Q) or negatively charged (R282D, R282E) substitutions gave a transporter that was not stimulated by external acidification (reducing pHout from 7.4 to 5.5) but transported at the same rate as the wild-type maximal rate (pHout 5.5); however, only the R282E mutation was unable to concentrate substrate above the extracellular level. All of the R282 mutants showed trans-stimulation of efflux comparable to the wild-type, except R282E-PepT1 which was faster. A conserved negatively charged residue, aspartate341 (D341) in transmembrane domain 8 was implicated in forming a charge pair with R282, as R282E/D341R- and R282D/D341R-PepT1 had wild-type transporter characteristics. Despite their differences in ability to accumulate substrate, both R282E- and R282D-PepT1 showed an increased charge:peptide stoichiometry over the wild-type 1:1 ratio for the neutral dipeptide Gly-l-Gln, measured using two-electrode voltage clamp. This extra charge movement was linked to substrate transport, as 4-aminobenzoic acid, which binds but is not translocated, did not induce membrane potential depolarisation in R282E-expressing oocytes. A model is proposed for the substrate binding/translocation process in PepT1.
1
Introduction
The proton-coupled di- and tri-peptide transporter PepT1 (SLC15a1) is the major route of uptake of dietary nitrogen from the intestine, and is also important along with the higher affinity gene product PepT2 (SLC15a2) in the re-absorption of filtered peptides in the kidney (Daniel & Kottra, 2004; Meredith & Boyd, 2000). In addition, PepT1 is the route of entry of a wide class of orally bio-available pharmaceutically important compounds, including the β-lactam antibiotics, angiotensin-converting enzyme (ACE) inhibitors, antiviral and anticancer agents (Terada & Inui, 2004). Although these therapeutic compounds are not di- or tri-peptides, they are carried by virtue of their similar 3D shape to endogenous substrates, i.e. they are peptidomimetic, and modelling of the substrate-binding site from the features in common of this huge and diverse range of substrates has led to predictions concerning which parts of the PepT1 protein may be important. For example, a substrate template model has been developed by several groups (Bailey et al., 2000; Bailey et al., 2006; Biegel et al., 2005) which allows prediction of binding affinity for a potential substrate.
The rabbit PepT1 is a 707 amino acid protein, with twelve transmembrane spanning domains (TMDs) as confirmed by epitope mapping (Covitz, Amidon, & Sadee, 1998). In the absence of a crystal structure, attempts have been made to computer model the PepT1 transporter itself (Bolger et al., 1998), with site-directed mutagenesis used to test hypotheses generated by these models. One potential complication for these kind of studies is our recent report that PepT1 may form multimers in the plasma membrane (Panitsas, Boyd, & Meredith, 2006) although it is not clear how the subunits interact.
Site-directed mutagenesis has been a useful tool to identify functionally important residues in PepT1. One such residue is arginine282 in the rabbit PepT1 sequence. The mutation of arginine282 to a glutamate produced a peptide transporter (R282E-PepT1) that was no longer driven by a proton-gradient but behaved more like a facilitated peptide transporter, whilst simultaneously exhibiting peptide-gated currents that were proposed to be through a non-specific cation channel activity (Meredith, 2004). Residue 282 is located approximately halfway down the predicted transmembrane domain 7 (TMD7), and is either an arginine or a lysine in all cloned mammalian PepT1 sequences to date. The presence of a charged amino acid residue in a TMD, along with its conservation, suggested a functional role. Here, we have systematically investigated the role of arginine282 in rabbit PepT1 by making further mutations to determine the requirement for the charge and have identified an interacting residue, aspartate341, located in putative TMD8. Some of these data have been previously published in abstract form (Pieri, Boyd, & Meredith, 2004).
2
Materials and methods
2.1
Site-directed mutagenesis of the PepT1 gene
Oligonucleotides were custom synthesised (Sigma-Genosys, UK) for the following sequences (residues in bold are changed from wild-type PepT1):-R282-PepT1 mutants forward:where xxx was CAA for R282Q, AAG for R282K, GAT for R282D, CAT for R282H, and GCG for R282A.-D341-PepT1 mutants forward:where xxx was CGC for D341R.
Reverse primers for the PepT1 mutant PCR reactions were the reverse compliment of the forward primers. The site-directed PepT1 mutants were generated using the Quikchange protocol (Stratagene), and the resulting constructs confirmed by DNA sequencing (Department of Biochemistry, University of Oxford, UK).
2.2
cRNA synthesis and oocyte injection
PepT1 constructs were linearised with XbaI (New England Biolabs, UK) and cRNA generated by in vitro transcription (T7 mMessage mMachine, Ambion, Cambridgeshire, UK). X. laevis oocytes were obtained under MS222 anaesthesia (0.2%, w/v) in accordance with the UK Animals (Scientific Procedures) Act, 1986, and maintained at 18 °C in modified Barth's medium (88 mM NaCl, 1 mM KCl, 0.82 mM MgSO4, 2.4 mM NaHCO3, 0.42 mM CaCl2, 10 mM Hepes, 5 mM sodium pyruvate, 50 μg ml−1 gentamicin (Fluka, Poole, UK), adjusted to pH 7.6 with 1 M NaOH). Transport measurements were performed at least 72 h after micro-injection of oocytes with 27nl cRNA (1 μg/μl), with medium changed daily.
2.3
Transport experiments
Zero-trans uptake of [3H]-d-Phe-l-Gln (17.4 Ci/mmole, custom synthesised, Cambridge Research Biochemicals, Stockton-on-Tees, UK) was performed as previously described (Meredith, 2004). Briefly, 5 oocytes were incubated in 100 μl of uptake medium (95 mM NaCl, 2 mM KCl, 1 mM CaCl2, 0.42 mM MgCl2, 10 mM Tris/Hepes pH 7.4 or Tris/Mes pH 5.5) with tracer (0.4 μM) [3H]-d-Phe-l-Gln. After incubation, the oocytes were washed sequentially five times in 1 ml of ice-cold 120 mM NaCl solution, lysed individually with 100 μl 2% (w/v) SDS and liquid scintillation counted. As a control non-injected oocytes were also incubated in uptake medium with [3H]-d-Phe-l-Gln as above.
The affinity of wild-type and mutant PepT1 were assessed by competition studies with 0.4 μM [3H]-d-Phe-l-Gln and Gly-l-Gln present in the uptake medium in concentrations from 0 to 2 mM using the protocol above, and the Ki calculated using the method of Deves and Boyd (1989).
Efflux studies were performed as previously described (Meredith, 2004), with the exception that the extracellular trans-stimulant Gly-l-Gln was used at 5 mM, and an efflux time-course was performed.
Diethylpyrocarbonate (DEPC) inhibition of PepT1 was performed using a similar protocol to that of Terada, Saito, and Inui (1998). Briefly, PepT1 oocytes were preincubated with 1 mM DEPC for 15 min at pHout 5.5 in the absence or presence of the PepT1 substrates Gly-l-Gln, N-Acetyl-Phe (Meredith et al., 2000), and l-Ala-Tyramine (custom synthesised) and the non-substrate Tyr (all 5 mM). The oocytes were then washed in uptake medium before uptake assays were performed as detailed above.
2.4
Electrophysiology
Measurements of membrane potential were made by impaling oocytes with a single glass microelectrode (Intra 767 amplifier, WPI, Stevenage, Hertfordshire, UK) perfused in uptake medium (as above) with or without 0.4 μM d-Phe-l-Gln (synthesised in house), 0.6 mM Gly-l-Gln (Sigma, Poole, UK) or 10 mM 4-aminobenzoic acid (4-AMBA, Sigma). Two-electrode voltage clamp (TEVC) was performed by placing oocytes in a 0.1 ml recording chamber and perfusing with uptake solution (pH 5.5 or 7.4) at a rate of 15 ml/min. Oocytes were impaled by two agarose-cushioned microelectrodes filled with 3 M KCl (0.5–2.0 MΩ) and voltage-clamped at −60 mV using a Geneclamp 500B amplifier and PCLAMP 8.1 software (Axon Instruments, CA, USA). The holding potential was stepped from −60 mV over the range of −150 to +50 mV in 10 mV steps, each pulse lasting 100 ms, and returning to −60 mV in between test voltage pulses. Typically traces were filtered at 1 kHz during recording and digitized at 0.5–5 kHz using the DigiData 1200 interface (Axon Instruments, CA, USA). All experiments were carried out at room temperature.
2.5
Data analysis
All data are expressed as mean ± S.E.M., except for Fig. 6 where the error bars represent the maximum range of stoichiometry values when taking into account the errors for the uptake data and the currents.
In order to calculate the apparent charge:substrate stoichiometry, the peptide induced current that was measured by two-electrode voltage clamp at the oocyte resting membrane potential was divided by the radiolabelled dipeptide uptake in oocytes from the same preparation. This value was normalised to 1:1 for wild-type PepT1, the accepted stoichiometry for a neutral dipeptide (Fei et al., 1994; Steel et al., 1997).
2.6
Statistical analysis
Statistical analyses were performed using one-way ANOVA with differences considered significant if p < 0.05 when data were compared to the wild-type control, as detailed in the text and/or figure legend.
3
Results
3.1
pH dependence of d-Phe-l-Gln uptake into R282 mutants
Fig. 1 shows the pH dependence of d-Phe-l-Gln uptake into oocytes expressing mutant PepT1 transporters where R282 has been changed into number of amino acid residues. Of the residues tested, R282K- and R282H-PepT1 behaved like the wild-type PepT1, in that the initial rate of uptake (1 h incubation time) of dipeptide was significantly faster (p < 0.05, one-way ANOVA) at an external pH of 5.5 than at 7.4. The other amino acid substitutions tested, R282E- (as previously reported, Meredith, 2004), R282D-, R282A- and R282Q-PepT1 all gave the same initial rate of uptake at pH 5.5 and 7.4, indicating that transport by these mutants is not stimulated by an inwardly directed proton gradient. These changes cannot be ascribed to changes in the affinity of the mutant PepT1 proteins for their substrate, as the Ki of Gly-l-Gln inhibiting 0.4 μM [3H]-d-Phe-l-Gln was unchanged in the mutants (Fig. 2).
3.2
Can the R282 mutant PepT1 transporters concentrate substrates?
An earlier finding was that, unlike the wild-type, the R282E-PepT1 mutant was unable to concentrate substrate even in the presence of an inwardly directed proton gradient (Meredith, 2004). The ability to concentrate substrate was therefore tested for the other R282 mutants (Fig. 3a and b shows representative time-course experiments at pHout 5.5 and 7.4, respectively), and the mean accumulation levels are shown for 8 h uptakes in Fig. 3c for pHout 5.5. In contrast to R282E-PepT1, all were found to be able to concentrate peptide well above the equilibrium level when the external pH was 5.5 (an oocyte was assumed to have a volume of 1 μl, Petersen & Berridge, 1996; Yao & Tsien, 1997). In the absence of the proton driving force (pHout 7.4, Fig. 3d) a similar level of intracellular d-Phe-l-Gln concentration was reached by all the mutant PepT1 transporters, including R282E-PepT1 if the incubation time was increased to 24 h (accumulation of 2.3 ± 0.4-fold compared to 3.1 ± 0.5-fold for wild-type PepT1). Although at 24 h incubation times we observed that cell survival can be a limiting factor, there was no statistical increase in the accumulation for the wild-type PepT1 at pHout 5.5 or 7.4 between 8 and 24 h incubations, nor between 8 and 24 h for the wild-type PepT1 at pHout 7.4 (8.0 ± 1.4 vs. 8.3 ± 1.0, 2.5 ± 0.5 vs. 3.1 ± 0.5 and 1.0 ± 0.4 vs. 1.5 ± 0.3 respectively, all p > 0.05, one-way ANOVA).
3.3
Rates of d-Phe-l-Gln efflux from R282 mutants
Fig. 4 shows the rates of efflux of [3H]-d-Phe-l-Gln from oocytes expressing either wild-type PepT1, R282 PepT1 mutants or non-injected controls. All of the PepT1 constructs showed a significantly faster efflux than the non-injected oocytes (one-way ANOVA, p < 0.05), whilst R282E-PepT1 was significantly faster than the wild-type (one-way ANOVA, p < 0.05) as previously described (Meredith, 2004). Interestingly, R282E-PepT1 showed a faster efflux than all the other R282 mutants (one-way ANOVA, p < 0.05), which were not significantly different to the wild-type (p = 0.14).
3.4
Apparent transport stoichiometry (charge to substrate) using two-electrode voltage clamp
As well as uptake being pH independent and non-concentrative, the electrophysiological characteristics of R282E-PepT1 were strikingly different to the wild-type (Meredith, 2004). Since R282D-PepT1 is similarly pH independent but does accumulate substrate, the apparent proton to peptide stoichiometry was examined, where the uptake of 0.4 μM d-Phe-l-Gln was compared to the peptide-induced current in the same batch of oocytes. The current at the oocyte resting membrane potential (−27.0 ± 1.1 mV at pHout 5.5, and −36.8 ± 2.2 mV at pHout 7.4, n = 12) was taken to represent the membrane potential under uptake conditions, as the addition of 0.4 μM substrate does not produce a detectable change in membrane potential (Fig. 5). The ratio of proton to neutral dipeptide co-transported through wild-type PepT1 is 1:1 (Fei et al., 1994; Steel et al., 1997), yet in R282E-, R282D- and R282A-PepT1 the apparent stoichiometry is substantially higher (4, 5 and 5 respectively at pHout 5.5, Fig. 6).
3.5
Is the extra current measured dependent on substrate transport?
In an attempt to see if the extra current carried by R282E-PepT1 was dependent on substrate translocation rather than simply substrate binding, the non-translocated PepT1 substrate 4-aminobenzoic acid (4-AMBA, Darcel, Liou, Tome, & Raybould, 2005; Meredith et al., 1998) was used. As found for wild-type PepT1, 4-AMBA failed to induce a depolarisation in R282E-PepT1 oocytes at 10 mM, three times its Ki (Meredith et al., 2000), in contrast to the known transported substrate Gly-l-Gln, also at three times Ki (Fig. 5).
3.6
Identification of an interacting residue for R282
The conservation of a positively charged amino acid in a transmembrane domain (TMD7), and the results above, strongly suggested that the presence of a positive charged residue was necessary for wild-type-like PepT1 transport function. In TMD8, predicted to be at approximately the same level in the membrane, there is a conserved aspartate (D341), and it was an appealing hypothesis that the two oppositely charged side chains might be forming a charge pair. To test this, double mutants were made, R282E/D341R- and R282D/D341R-PepT1, to swap the charges over. As can be seen in Fig. 7, these double mutants showed the same pH dependence of influx as the wild-type transporter, providing strong evidence to support the hypothesis. Both R282E/D341R- and R282D/D341R-PepT1 were also able to concentrate substrate like the wild-type (data not shown).
3.7
Diethylpyrocarbonate inhibition of PepT1 function
Preincubation of wild-type PepT1-expressing oocytes with 1 mM diethylpyrocarbonate (DEPC) for 15 min completely inhibited the PepT1 mediated dipeptide uptake measured over 1 h, as shown in Fig. 8. This inhibition by DEPC was largely prevented by the presence of the known PepT1 substrates Gly-l-Gln and N-Acetyl-Phe (Meredith et al., 2000), but not by the amino acid non-substrate Tyr. Interestingly, despite being a good PepT1 substrate (Ki approximately 0.1 mM, data not shown), a modified peptide lacking a carboxyl terminus (l-Ala-Tyramine) only partially prevented DEPC inhibition (Fig. 8).
4
Discussion
The R282K mutation of rabbit PepT1 is not only the most conservative one regarding the charge, but in a number of species, including dog, rat and mouse, lysine is the naturally occurring residue at this position. Therefore it was not surprising to find that this mutant behaves like the wild-type rabbit PepT1 (pig, sheep, rhesus and crab-eating monkeys and human also have R282). The finding that R282H also behaved like the wild-type was interesting, as histidine has a side-chain that can be titrated over the range used in the experiments (pK of ∼6 in free solution). Our findings could be interpreted in several ways, including the possibility that only at pHout 5.5 is there the formation of a positive charge by side-chain titration that gives a stimulation of uptake over that seen at pHout 7.4. A second possibility is that the protein environment surrounding H282 is such that its side chain pK is shifted away from 6 and it is therefore always protonated, and thus behaves more like an arginine or lysine. This effect has been shown for example in the enzyme protein tyrosine kinase (Tishmack, Bashford, Harms, & Van Etten, 1997), where histidine residues had a pK as high as 9.2 when analysed by NMR.
In the original study on R282E-PepT1, it was concluded that the uptake of peptide by the mutant transporter was uncoupled from the movement of protons, and that in addition to acting as a facilitated peptide transporter, R282E-PepT1 also displayed a peptide-gated non-specific cation conductance (Meredith, 2004). However, it is possible that this conclusion needs updating in the light of the current findings that there are R282 mutants that, like R282E-PepT1, are not pH stimulated, yet are still able to accumulate substrate above equilibrium when an inwardly directed proton gradient is imposed. For a transporter to be able to accumulate substrate above equilibrium, an energy source must be involved, in this case the proton electrochemical gradient. Therefore, the mutant PepT1 proteins that can accumulate substrate but do not show pH stimulation (R282A and R282D) must still be coupled to the movement of protons down their electrochemical gradient. The lack of pH stimulation could be attributed to the fact that during the transport cycle these specific mutants have a different rate limiting step to the wild-type, and that for these mutants that step is not pH dependent. It has already been reported that R282E-PepT1 has a faster rate of efflux than wild-type, consistent with an uncoupling of peptide uptake from the proton driving force (Meredith, 2004); the finding here that the rates of efflux for the other R282 mutants are not different from that of the wild-type is in agreement with the hypothesis that they are still proton-coupled, as shown by their ability to accumulate substrate above the extracellular concentration at pHout 5.5 but not 7.4.
The simplest hypothesis was that in R282E-PepT1, the extra inward charge movement associated with peptide uptake collapsed the membrane potential, which is known to be the major driving force for proton-coupled peptide uptake (Temple & Boyd, 1998), hence the apparent lack of substrate accumulation. Therefore by extension one would expect R282D- and R282A-PepT1 to have the same charge coupling as the wild-type, as they too accumulate substrate, but this was not the case: both R282E-, R282D- and R282A-PepT1 showed the same increased charge:peptide apparent stoichiometry, i.e. substantially larger than the wild-type. This stoichiometry itself showed pH dependence, with a lower value of around 2 for the mutants at pH 7.4, suggesting that the current is either carried by protons or is a pH-sensitive phenomenon. There was no difference in the uptake in the absence or presence of sodium, either for R282E-PepT1 (Meredith, 2004) or R282D-PepT1 (data not shown).
The finding that the R282E/D341R- and R282D/D341R-PepT1 double mutants (the latter being a charge pair reversal of the naturally occurring residues in rabbit PepT1) had the same characteristics as the wild-type protein strongly suggests that these two residues do interact in the 3D protein (Pieri et al., 2004) as previously proposed (Meredith, 2004). During the preparation of this manuscript, Kulkarni et al. (2007) reported findings consistent with R282 and D341 forming a charge pair in human PepT1. The initial hypothesis that in R282E-PepT1 repulsion between E282 and D341 allowed the movement of extra ions through the protein when a peptide is transported was not supported however by the finding that the single D341R mutant also behaved like the wild-type, as one might have thought that R282 and R341 would repel in much the same way as E282 and D341. That D341R-PepT1 behaved like wild-type suggests that although R282 and D341 seem to form a charge pair, in rabbit PepT1 residue 341 being negatively charged is not crucial to PepT1 function; interestingly, in human PepT1 the D341R single mutant had reduced function (Kulkarni et al., 2007). The reason for this difference between rabbit and human PepT1 is not clear. The observation that the non-transported PepT1 substrate 4-AMBA did not induce a depolarisation in R282E-PepT1-expressing oocytes clearly shows that the charge movement is linked to substrate binding and translocation and not to binding alone. One explanation for the increase in proton–peptide stoichiometry is that in wild-type PepT1 the presence of a positively charged residue deep in the binding pocket at position 282 repels proton movement through the transporter protein during the translocation step. The data in Fig. 6 are consistent with this, as the apparent stoichiometry is lower at pHout 7.4 than it is at pHout 5.5, indicating that the proton electrochemical gradient is involved.
In the case of mutations where R282 was replaced with a non-positively charged amino acid, the rate-limiting step of the transport cycle must be insensitive to extracellular pH, hence the lack of stimulation when the pHout is dropped from 7.4 to 5.5. Kinetic analysis of peptide transport by PepT1 in rat renal brush border membrane vesicles (Temple & Boyd, 1998) showed that at pHout7.4/pHin7.4, it was the protonation of the carrier protein that was the rate-limiting step (Temple, Bailey, Bronk, & Boyd, 1996), whereas at pHout5.5/pHin7.4 it was the return of the unloaded carrier. The rate of peptide uptake by R282E-PepT1 (corrected for protein expression in the intact oocyte plasma membrane by luminometry, Panitsas et al., 2006) is the same as for the wild-type at pHout 5.5 (data not shown), but, unlike for the wild-type, is not slower at pHout 7.4. This indicates that the mutations to arginine282 that abolish pH sensitivity (R to E, D, A, or Q) are affecting the rate limiting step, so when pHout is 7.4 the rate limiting step has the same magnitude as that at pHout 5.5, hence the lack of sensitivity to changing pHout. The rate limiting protonation of the outward-facing carrier protein at pHout 7.4, the first step in the transport cycle (Temple et al., 1996), was proposed by us (Bailey et al., 2000; Meredith & Boyd, 1995) and others (e.g. Steel et al., 1997; Uchiyama, Kulkarni, Davies, & Lee, 2003) to be protonation of histidine57 (H57). In the R282-PepT1 mutants, except for R282K and R282H, the lack of a positively charged residue might result in a conformational change of the protein that changes the local environment and increases the pKa of H57, such that it is more easily protonated at a higher pHout. Thus at pHout 7.4 the rate limiting step is no longer the protonation of H57, but the return of the unloaded carrier, as it is at pHout 5.5, hence the similar transport rates at pHout 5.5 and 7.4.
A model for how proton–peptide transport might occur is shown in Fig. 9: the empty PepT1 is primed by protonation of H57, followed by the binding of a zwitterionic peptide, with the N-terminal co-ordinated at E595 and the C-terminal at H57-H+ (Meredith et al., 2000). Although the binding of the substrate C-terminal to His57-H+ is in disagreement with the conclusion of Terada et al. (1998), it is supported by the finding that N-Acetyl-Phe, a known PepT1 substrate which does not have a free amino terminal (Meredith et al., 2000), can protect PepT1 against inhibition by DEPC. Additionally, the finding that significant DEPC inhibition is still evident when oocytes were co-incubated with the PepT1 substrate l-Ala-Tyramine which lacks a carboxyl terminus further adds to the notion that H57-H+ binds the carboxyl terminus of the substrate (Fig. 8). H57-H+ then donates its proton to the C-terminal carboxyl group, and the transporter undergoes a conformational change that leads to the breaking of the salt bridge between R282 and D341, re-orientating the binding site to be inward facing as simultaneously the protonated N-terminal of the substrate binds to D341, neutralising the charge, and the R282 charge is stabilised by Y167 (the chemical properties of this tyrosine have been shown to be essential, Yeung et al., 1998). The peptide molecule is then released into the cytoplasm, whereby it returns to the zwitterionic state by releasing the proton from the carboxyl terminal. The transporter then undergoes the reverse conformational change to re-orientate the binding site to outward facing, and R282 reforms the salt bridge with D341.
As R282E-PepT1 cannot accumulate substrate, the implication must be that the movement of peptide is no longer coupled to the movement of the protons, whereas in all the other mutants coupling must be maintained. If for R282E-PepT1 the pKa of H57 was raised to the point that it was no longer favourable for it to donate its proton to the carboxyl terminus of the substrate, then peptide transport would no longer be proton-coupled, and this would explain the failure of R282E-PepT1 to accumulate substrate. Intriguingly, it can be seen in Fig. 6 that both R282D- and R282A-PepT1 appear to carry approximately one more charge per substrate peptide than R282E-PepT1, which is consistent with the hypothesis that one of the charges carried is coupled with the substrate.
In conclusion, the arginine at position 282 in rabbit PepT1 plays an intriguing role in the function of the transporter, with mutations to different residues revealing that a positive residue is required for pH dependence, whilst only R282E-PepT1 cannot concentrate substrate above equilibrium; this is despite other mutations, most notably R282D-PepT1, having a similarly increased charge:peptide stoichiometry. As previously proposed, R282 (TMD7) forms a charge pair with D341 (TMD8), with R282E/D341R-PepT1 showing normal transport characteristics. Further biological testing or a crystal structure of PepT1 will be required to establish the validity of the model proposed. | [
"site-directed mutagenesis",
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Exp_Brain_Res-4-1-2373863 | Intramanual and intermanual transfer of the curvature aftereffect
| The existence and transfer of a haptic curvature aftereffect was investigated to obtain a greater insight into neural representation of shape. The haptic curvature aftereffect is the phenomenon whereby a flat surface is judged concave if the preceding touched stimulus was convex and vice versa. Single fingers were used to touch the subsequently presented stimuli. A substantial aftereffect was found when the adaptation surface and the test surface were touched by the same finger. Furthermore, a partial, but significant transfer of the aftereffect was demonstrated between fingers of the same hand and between fingers of both the hands. These results provide evidence that curvature information is not only represented at a level that is directly connected to the mechanoreceptors of individual fingers but is also represented at a stage in the somatosensory cortex shared by the fingers of both the hands.
Introduction
The neural representation of haptic information can be investigated using different approaches. The representation of object shape perceived with the fingers has mainly been studied using neurophysiological tools. It has been found that especially slowly adapting type I (SAI) mechanoreceptors in the finger but also fast-adapting type I (FAI) receptors are sensitive to curvature (Goodwin et al. 1997; Jenmalm et al. 2003). In order to perceive curvature, a combination of responses from a population of receptors is required (Goodwin and Wheat 2004). This processing occurs along several stages up to at least the somatosensory cortex (SI) (Gardner and Kandel 2000). Taking a neurophysiological approach is useful to uncover the pathways underlying curvature processing, but is less appropriate to establish the levels at which perceived curvature is essentially represented.
A psychophysical approach that has been successful in providing greater insight into the neural representation of perceived properties is the study of the aftereffect, and especially, the transfer of the aftereffect. In vision, for example, the finding of partial, interocular transfer of the motion aftereffect has been explained by the involvement of both monocular and binocular cells in the processing of motion information from the stimulus (Moulden 1980; Wade et al. 1993; Tao et al. 2003). In a similar way, establishing the transfer characteristics of a haptic curvature aftereffect would provide insight into the representation of shape information. Finding aftereffect transfer between different fingers would indicate that curvature is represented at a level shared by these fingers, whereas no transfer would imply that each finger has a separate representation of curvature.
A curvature aftereffect is the phenomenon whereby a flat test surface feels concave following prolonged contact with a convex adaptation surface (see Fig. 1a). Curvature aftereffects have been found for different shapes and exploration modes. Gibson (1933) reported that a flat cardboard edge felt concave after the prolonged dynamic exploration of a convex cardboard edge. Vogels et al. (1996) demonstrated the existence of an aftereffect when the whole hand was placed on spherically curved shapes. They performed extensive experiments to examine the characteristics of this static curvature aftereffect. They found a linear relationship between the magnitude of the aftereffect and the curvature of the adaptation stimulus. Furthermore, they showed that the magnitude of the aftereffect increased with the adaptation time up to about 10 s. Finally, they found a decrease of the aftereffect with an increase of the interstimulus interval. In a follow-up study, they showed that the aftereffect also existed for alternative exploration modes, like touching a stimulus with only the five fingertips of the hand or performing small movements of the hand over the stimulus surface (Vogels et al. 1997). Given the strength and consistency of these findings, we supposed that curvature aftereffects should also occur for alternative ways of touching, such as the situation in which curved surfaces are statically being touched with only a single fingertip. However, this phenomenon has not yet been investigated, and consequently, any curvature aftereffect transfer between the fingers also remains unexplored.Fig. 1a Schematic overview of a haptic curvature aftereffect: when you first touch a convex (concave) surface for some time, say 10 s, and subsequently touch a flat surface, this latter surface feels concave (convex). b Schematic drawings of the cross-sections of a convex and a concave stimulus. The stimuli had a cylindrical shape with a spherical top (see illustration a). The distance from the bottom to the centre of the top (h) was consistently 30 mm. The diameter of the cylinders (d) was also 30 mm. c Examples of two psychometric curves. The circular data points and the fit through these points results from adaptation to the convex adaptation stimulus. The PSE is represented by PV. The square data points and the fit through these points result from adaptation to the concave adaptation stimulus. In this case, the PSE is represented by PC. The magnitude of the aftereffect (AE) is defined as the difference between PV and PC
The purpose of the present study was to obtain a better understanding of the representation of haptically perceived shape information, by probing the transfer of the curvature aftereffect. In the first experiment, we established the existence of an aftereffect when a curved surface is touched by a single finger and measured whether this aftereffect transferred to other fingers of the same hand. The second experiment was set up to determine whether the aftereffect depended on the finger used. Finally, in the third and fourth experiments, we investigated the transfer of the aftereffect between fingers of both hands.
Materials and methods
Subjects
A total number of 40 subjects participated [n = 8 for experiments 1, 2 and 4, n = 16 for experiment 3; 18 were male and 22 were female; the mean age was 22 years; 37 were right-handed, 3 were left-handed, according to a standard questionnaire (Coren 1993)]. Subjects in experiments 1 and 2 received course credit for their participation. Subjects in the third and fourth experiments received monetary compensation.
Stimuli
The stimuli comprised of a compound of polyurethane foam and artificial resin (Cibatool BM 5460). A computer-controlled milling machine was used to produce cylinders with a flat bottom and a spherically curved top. The top was either pointing outward (convex) or inward (concave). Both convex and concave adaptation stimuli were used, with curvature values of +36 and −36 m−1, respectively; the curvature of the nine test stimuli ranged from −16 to +16 m−1, in steps of 4 m−1. Illustrations of the stimuli and their cross-sections are given in Fig. 1a, b, respectively.
Procedure
A subject was seated behind a table. The preferred arm rested on a platform, which was 30 mm above the tabletop. In the third and fourth experiments, both arms rested on the platform. Only the fingertips projected over the platform. The experimenter placed the stimulus underneath a fingertip. A curtain prevented the subjects from seeing the stimulus. During a trial, the tip of one finger was placed on an adaptation stimulus for 10 s. Subsequently, the subject placed a finger on a test stimulus and had to judge whether this test stimulus felt convex or concave. Subjects were not allowed to move the finger over the stimulus surface, and the experimenter checked for this. No instructions were given on the force to contact the stimulus, nor was it measured. No feedback was provided on the response.
Three conditions were measured in the first experiment. In all conditions, the adaptation stimulus was touched with the index finger. In one condition, the test stimulus was also touched with the index finger. In the other two conditions, the test stimulus was touched with the middle finger or the little finger of the same hand, respectively. Each condition consisted of 10 repetitions of a group of 18 trials (two adaptation stimuli × nine test stimuli) with trials randomized within a group. One complete condition was measured in a single session of about one and a half hours. The separate sessions were spread over different days. The order in which the conditions were conducted was counterbalanced for the first six subjects and randomly chosen for the last two subjects.
In the second experiment, both the adaptation and the test stimuli were touched by the middle finger. In the third and fourth experiments, the adaptation stimulus was contacted by the index finger of the preferred hand; the test stimuli were touched with the index finger (third experiment) or middle finger (fourth experiment) of the non-preferred hand.
Analysis
The data for each subject and each condition were analyzed separately for the convex and the concave adaptation stimuli. The percentage of “convex” responses was plotted against the curvature of the test stimulus. The point of subjective equality (PSE) was determined by fitting a psychometric function (cumulative Gaussian) to the data. The PSE represents the curvature value that in 50% of the test cases was judged “convex” and in 50% of the cases was judged “concave”. The magnitude of the aftereffect is defined as the difference between the PSE resulting from the adaptation to a convex surface and the PSE resulting from the adaptation to a concave surface. Examples of psychometric curves for a convex and a concave adaptation are given in Fig. 1c.
Results
The mean results for the aftereffect values are shown in Fig. 2. The error bars indicate the standard errors of the mean.Fig. 2Mean results of the aftereffect. The indicated error bars are the standard error in the mean for each condition. Experiment 1: eight subjects participated. Adaptation was performed by the index finger of the preferred hand. Testing was done using the index finger, middle finger, or little finger of the same hand. Experiment 2: eight subjects participated. Adaptation and testing was performed by the middle finger of the preferred hand. Experiment 3: sixteen subjects participated. Adaptation was performed by the index finger of the preferred hand; testing was done by the opposite index finger. Experiment 4: eight subjects participated. Adaptation was performed by the index finger of the preferred hand; testing was done by the middle finger of the non-preferred hand
Experiment 1
We tested the occurrence of an aftereffect in each condition by performing separate one-tailed t tests. A significant result was obtained in all conditions (t7 = 6.3, P < 0.001 for the index finger; t7 = 9.8, P < 0.001 for the middle finger; t7 = 3.4, P = 0.006 for the little finger). Subsequently, an ANOVA with a repeated measures design was performed to determine any differences between conditions. A significant main effect was found (F2,14 = 22.5, P < 0.001). Pairwise comparisons showed a significant difference between the index finger and the middle finger (P = 0.007) and between the index finger and the little finger (P = 0.004), but not between the middle finger and the little finger (P = 1.0). The P values were adjusted with a Bonferroni correction.
Experiment 2
A one-tailed t test showed that there was a significant aftereffect (t7 = 8.0, P < 0.001). Inspection of Fig. 2 shows that the aftereffect of the middle finger condition of the second experiment was comparable to the index finger condition of the first experiment and was much higher than the middle finger condition of the first experiment. Independent samples t test confirmed that there was no significant difference in the first case (t14 = 0.6, P = 0.6), but that there was a significant difference in the second case (t7.4 = 6.1, P < 0.001).
Experiment 3
A one-tailed t test highlighted a significant aftereffect (t15 = 2.7, P = 0.009). The magnitude of this aftereffect was much lower than for the index finger condition of the first experiment. This was confirmed by an independent sample t test (t22 = 5.0, P < 0.001).
Experiment 4
A significant aftereffect was obtained, as was confirmed by a one-tailed t test (t7 = 7.4, P < 0.001).
Discussion
The first novel observation of this paper is that the perception of surface curvature by a single fingertip is influenced by preceding contact of this finger with another curved surface. The magnitude of this curvature aftereffect did not depend on the finger employed, as shown by a comparison between the results of the first and the second experiment. Previously, Vogels et al. (1996, 1997) reported the existence of a static curvature aftereffect, when the whole hand was used. We suppose that our finding of a one-finger aftereffect falls in the same class of phenomena. A quantitative comparison between the results of Vogels et al. (1996) and our finding can be made by calculating the relative magnitude of the aftereffect, i.e. the aftereffect divided by the difference between the adaptation stimuli. This value equals 0.17 ± 0.02 for the results of Vogels et al., whereas it was 0.15 ± 0.07 for the index finger condition of the first experiment and 0.17 ± 0.06 for the middle finger condition of the second experiment, respectively. These values are in the same order of magnitude, irrespective of the differences in manner of touching and curvature range of the stimuli.
The second important finding of our study is that the aftereffect partially transfers between fingers of the same hand. This means that the sensation of shape with a certain finger influences the perception of a shape touched by another finger. This suggests that the sensations obtained by the different fingers share a common representation. However, the transfer is far from complete, indicating that curvature perception by each finger also yields a substantial, individual part in the representation. Interestingly, the aftereffect does not only transfer from the index finger to the neighboring middle finger, but also to the distant little finger. This result is unlike recently performed localization (Schweizer et al. 2000) and learning studies (Sathian and Zangaladze 1997; Harris et al. 2001), in which the reported transfer effects were obtained in the neighboring finger, but not in the distant fingers. This indicates that the processes involved in detecting the finger that is stimulated or increasing the skills to discriminate punctate pressure or roughness are quite different from those concerned in shape perception of an object.
The third interesting result of this study is that there was a small, but significant transfer of the aftereffect between fingers of both hands, irrespective of whether opposite fingers (experiment 3) or different fingers (experiment 4) were employed. This result is different from the result reported by Vogels et al. 1997, who did not find intermanual transfer. However, in their experiments, whole hands were involved, whereas only single fingertips were used in our experiment. Moreover, their conclusion was based on the performance of only 2 subjects, whereas 24 participants provided the data for our study. The results of the third and fourth experiments suggest that the representation of shape information obtained with one hand is not completely distinct from the representation of shape information received by the other hand, but shares a common, bilateral component.
How can our findings be interpreted in the context of neurophysiological literature? Firstly, our finding that the aftereffect only transfers partially between fingers of the same hand shows that a substantial part of the processing occurs at a stage where each finger is individually represented. On this stage, which spreads from the mechanoreceptors in the fingers up to area 3b in SI, no overlap occurs in signals from the slowly adapting receptors and the fast-adapting receptors (Gardner and Kandel 2000). Slowly adapting receptors respond with a sustained discharge when the finger is in contact with a surface, whereas fast-adapting receptors only respond at the onset and removal phase of the finger (Johansson and Vallbo 1983). Vogels et al. (1996) showed that the magnitude of their curvature aftereffect increased with an increase in adaptation time. These findings point to an important role for the slowly adapting receptors in the curvature aftereffect. Therefore, we suggest that the aftereffect at the stage related to an individual finger mainly originates from the processing of the slowly adapting receptors. Secondly, the fact that we found a transfer between the fingers of the same hand implies that a significant part of the processing of curvature information occurs at a level shared by the different fingers. In physiological terms, this indicates that at least area 1 or 2 of SI are involved, as receptive fields in these areas cover several fingers of a single hand (Gardner and Kandel 2000), but processing may also occur at an even higher stage. Thirdly, our finding of an intermanual transfer shows that the processing of curvature information also takes place on a higher, bilateral level. We can only speculate on the neural correlates of this bilateral processing. Possible candidates include area 2 of SI, areas 5 and 7 of the posterior parietal cortex, and the secondary somatosensory cortex (Iwamura 2000; Gardner and Kandel 2000).
It is interesting to mention that the aftereffects in the intramanual transfer conditions (experiment 1) and the intermanual transfer conditions (experiment 3 and 4) are similar in magnitude. This suggests that no important curvature processing occurs at a level that is devoted to a single hand, but that all processing takes place at a higher stage. The similar results for experiments 3 and 4 provide further support that the hands and fingers are not somatotopically represented at this stage. From a previous study, it is known that subjects also performed similarly in intramanual and intermanual curvature discrimination tasks, but that higher performance was obtained when only a single finger was employed (Van der Horst and Kappers 2007). The analogy between that study and the current study is that curvature information is mainly represented at the level of the individual finger, but partly available at a higher, finger- and hand-independent level. We should be careful in ascribing a specific function to the involvement of the higher level areas in the processing of curvature information. The role of more cognitive aspects should not be excluded, since it is known that processes like tactile attention (Burton and Sinclair 2000; Spence and Gallace 2007), working memory (Burton and Sinclair 2000), and object recognition (Reed et al. 2004) also engage the somatosensory areas.
The aftereffect that we found in the present study is a similar phenomenon as the aftereffect that was previously reported by Vogels et al. (1996, 1997). However, this does not entail that the representation of curvature is identical for touching with a single finger or with the whole hand. Vogels et al. (1997) already showed that, although similar aftereffects were found when either the whole hand or only the five fingertips were used, only a small transfer between these exploration modes was obtained, which points to a limited overlap in representation. Similarly, we suppose that there is a difference in representation between curvature that is perceived by a single finger and curvature that is perceived by the whole hand. In the single finger case, the representation is mainly at the level of the individual finger, whereas in the whole hand case, the representation is spread over all fingers and the palm of the hand.
This study shows that establishing the intramanual and intermanual transfer of the aftereffect is a useful tool in obtaining more insight into the representation of object properties as perceived by the fingers. In general, studying aftereffect transfer is attractive, because it enables a connection between psychophysics and neurophysiology. The convergence of these approaches leads to a better understanding of human perception. | [
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Eur_J_Appl_Physiol-3-1-1914221 | Physical fitness, fatigue, and quality of life after liver transplantation
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Clin_Rev_Allergy_Immunol-4-1-2243253 | "Anti-CCP2 Antibodies: An Overview and Perspective of the Diagnostic Abilities of this Serological M(...TRUNCATED) | "The literature of the last 4 years confirms that the anti-CCP2 test is a very useful marker for the(...TRUNCATED) | [
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Schutz 2008 PubMed dataset for keyphrase extraction
About
This dataset is made of 1320 articles with full text and author assigned keyphrases.
Details about the dataset can be found in the original paper: Keyphrase extraction from single documents in the open domain exploiting linguistic and statistical methods. Alexander Thorsten Schutz. Master's thesis, National University of Ireland (2008).
Reference (indexer-assigned) keyphrases are also categorized under the PRMU (Present-Reordered-Mixed-Unseen) scheme as proposed in the following paper:
- Florian Boudin and Ygor Gallina. 2021. Redefining Absent Keyphrases and their Effect on Retrieval Effectiveness. In Proceedings of the 2021 Conference of the North American Chapter of the Association for Computational Linguistics: Human Language Technologies, pages 4185–4193, Online. Association for Computational Linguistics.
Text pre-processing (tokenization) is carried out using spacy (en_core_web_sm model) with a special rule to avoid splitting words with hyphens (e.g. graph-based is kept as one token). Stemming (Porter's stemmer implementation provided in nltk) is applied before reference keyphrases are matched against the source text.
Content
The details of the dataset are in the table below:
| Split | # documents | # keyphrases by document (average) | % Present | % Reordered | % Mixed | % Unseen |
|---|---|---|---|---|---|---|
| Test | 1320 | 5.40 | 84.54 | 9.14 | 3.84 | 2.47 |
The following data fields are available:
- id: unique identifier of the document.
- title: title of the document.
- text: full article minus the title.
- keyphrases: list of reference keyphrases.
- prmu: list of Present-Reordered-Mixed-Unseen categories for reference keyphrases.
NB: The present keyphrases (represented by the "P" label in the PRMU column) are sorted by their apparition order in the text (title + text).
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